Metal ion complexes

ABSTRACT

The present invention provides a process for the preparation of metal ion complexes, the process comprising contacting a metal in the form of particles with a chelating agent in solid form and, while the metal is in contact with the chelating agent, contacting the metal and chelating agent with an oxidising agent. The present invention also provides novel metal ion complexes. The invention further provides uses of the metal ion complexes.

FIELD OF THE INVENTION

The present invention relates to metal ion complexes, a process for thepreparation of metal ion complexes, compositions comprising metal ioncomplexes and the uses thereof.

BACKGROUND

Metals, metal ions and their complexes play an important physiologicalrole in animals, humans and plants. For example, metals such as copper,zinc, iron, calcium and the like, are important for, among other things,bone development, energy production and fatty acid oxidation. Inaddition, metals may be incorporated into enzymes and proteins, and mayalso function as co-factors (for example, copper is a co-factorassociated with the enzyme superoxide dismutase).

Administration of metal ions and their complexes can be therapeuticallyuseful. For example, metal ions, such as copper ions, have beenadministered in the form of salts, such as, for example, metal sulfides,chlorides and nitrates, for various therapeutic uses.

The specification for Australian patent application no. 2004205086 (AU2004205086 A1) discloses a process for preparing a metal ion chelatecomplex. This document discloses a process for preparing copper EDTA andrelated complexes, involving mixing a chelating agent with an oxidisingagent in solution to obtain a mixture and adding at least one metalcapable of forming a metal ion chelate complex with said chelating agentto said mixture, so as to form at least one metal ion chelate complex,or a mixture thereof.

It would be advantageous to provide metal ion complexes with differentand/or new structures. It would also be advantageous to providealternative processes for preparing metal ion complexes.

SUMMARY OF THE INVENTION

The present inventors have developed a novel process for preparing metalion complexes, novel metal ion complexes, and methods of using suchcomplexes.

In a first aspect, the present invention provides a process for thepreparation of a metal ion complex, the process comprising:

-   -   (a) contacting a metal in the form of particles having a bulk        density of between about 0.2 and about 8.0 g/cm³ with a        chelating agent in solid form; and    -   (b) while the metal is in contact with the chelating agent,        contacting the metal and chelating agent with an oxidising        agent, to form at least one metal ion complex.

The process typically comprises the further step (c) of allowing thecombination of agents resulting from step (b) to react until completion,i.e. allowing the oxidising agent, metal and chelating agent to reactuntil completion.

The inventors have surprisingly found that metal ion complexes havingdifferent properties to metal ion complexes prepared by prior artprocesses can be prepared using the process of the present invention andallowing the reaction to proceed until completion.

For example, processes described in the Examples of AU 2004205086 A1afford the metal ion chelate complex Cu[EDTA]²⁻. When copper and EDTAare used in the process of the present invention, and the reactionallowed to proceed to completion, a copper ion complex having differentproperties to Cu[EDTA]²⁻ can be prepared.

In an embodiment, the bulk density of the metal is between about 0.3 andabout 4.0 g/cm³. In an embodiment, the bulk density of the metal isbetween about 0.8 and about 2.5 g/cm³.

In an embodiment, the metal and chelating agent are above ambienttemperature when the metal and chelating agent are contacted with theoxidising agent.

In an embodiment, the oxidising agent is above ambient temperature whenthe metal and chelating agent are contacted with the oxidising agent.

In an embodiment, the ratio of metal:chelating agent in moles is withinthe range of about 1:1 to 50:1.

In an embodiment, the ratio of chelating agent:oxidising agent in molesis within the range of about 1:1 to 1:20.

In an embodiment, the metal is selected from the group consisting of Cu,Zn, Mn, Fe, Co, Ni, Cr, Al, Cd, Ag, Au, Se, and mixtures thereof.

In an embodiment, the metal is Cu.

In an embodiment, the chelating agent comprises a nitrogen and/or anoxygen donor.

In an embodiment, the chelating agent is selected from the groupconsisting of ethylenediaminetetraacetic acid (EDTA),diethylenetriaminepentaacetic acid (DTPA),hydroxyethylenediaminetetraacetic acid (HEDTA),ethylenediaminedisuccinic acid (EDDS), salicylic acid, acetyl salicylicacid, amino acids, peptides, and salts and hydrates thereof. In anembodiment, the amino acid is selected from the group consisting ofglycine, histidine, lysine, arginine, cysteine, methionine and saltsthereof, or mixtures thereof.

In an embodiment, the chelating agent is ethylenediaminetetraacetic acid(EDTA) or a salt thereof.

In an embodiment, the chelating agent is ethylenediaminetetraacetic acid(EDTA) disodium salt.

In an embodiment, the oxidising agent is selected from the groupconsisting of peroxides, peracids, ozone and oxidising salts.

In an embodiment, the oxidising agent is in solution when the metal andchelating agent are contacted with the oxidising agent.

In an embodiment, the oxidising agent is in aqueous solution when themetal and chelating agent are contacted with the oxidising agent.

In an embodiment, the peroxide is hydrogen peroxide.

In an embodiment, the hydrogen peroxide is in an aqueous solution.

In an embodiment, the aqueous solution of hydrogen peroxide has aconcentration in the range of about 5 wt. % to about 60 wt. %.

In an embodiment, the aqueous solution of hydrogen peroxide has aconcentration of about 50 wt. %.

In an embodiment, the metal is Cu, the chelating agent isethylenediaminetetraacetic acid (EDTA) disodium salt and the oxidisingagent is hydrogen peroxide.

In an embodiment, hydrogen peroxide is in an aqueous solution, the ratioof Cu:ethylenediaminetetraacetic acid (EDTA) disodium salt in moles isabout 1:1 to about 50:1 and the ratio of Cu:hydrogen peroxide in molesis about 1:1 to about 1:20.

In an embodiment, Cu in contact with ethylenediaminetetraacetic acid(EDTA) disodium salt is maintained at above about 75° C. (e.g. about 80°C. to 90° C.) when the Cu and EDTA are contacted with hydrogen peroxide,the hydrogen peroxide also being at above about 75° C. (e.g. about 80°C. to 90° C.).

In an embodiment, the process further comprises a step of removingunreacted metal and/or unreacted chelating agent and/or unreactedoxidising agent.

In a second aspect, the present invention provides a metal ion complexobtained by the process according to the first aspect.

In a third aspect, the present invention provides a compositioncomprising a metal ion complex or a mixture of metal ion complexesobtained by the process according to the first aspect.

In a fourth aspect, the present invention provides a formulation fortopical administration comprising a metal ion complex or a mixture ofmetal ion complexes obtained by the process according to the firstaspect.

In a fifth aspect, the present invention provides the use of a metal ioncomplex or a mixture of metal ion complexes obtained by the processaccording to the first aspect in the manufacture of a medicament forreducing inflammation, treating or preventing inflammation associatedwith arthritis, promoting hair growth, treating or preventing psoriasis,increasing blood flow, treating or preventing chilblains, treating orpreventing varicose veins, treating a wound, promoting wound healing,treating or promoting healing of scar tissue, treating burns,alleviating or reducing joint pain, alleviating or reducing muscularpain, alleviating or reducing neuropathic or nerve pain, treating orpreventing sinus inflammation, treating or preventing sinus pain,treating or preventing a bacterial infection, treating or preventing afungal infection, treating or preventing eczema, treating or preventingwrinkles, treating or preventing bruises, treating or preventing jointdegeneration, treating or preventing cartilage degeneration, treating orpreventing acne, treating or preventing muscle damage, treating orpreventing motor neuron disease (MND; MND includes, for example,amyotrophic lateral sclerosis (ALS; also known as Lou Gehrig's Disease),primary lateral sclerosis (PLS), progressive muscular atrophy (PMA),progressive bulbar palsy (PBP) and pseudobulbar palsy), treating orpreventing viral conditions, treating or preventing prion diseases (e.g.bovine spongiform encephalopathy (BSE), scrapie, Creutzfeldt-Jakobdisease (CJD) or its variant (vCJD), Gerstmann-Straussler-Scheinkersyndrome, fatal familial insomnia or kuru), treating or preventing jointdamage, treating or preventing tendon damage, treating or preventingonychoschizia (split nails), treating or preventing cancer, treating orpreventing breast cancer, treating or preventing brain cancer, treatingor preventing melanoma, treating or preventing basal cell carcinoma,treating or preventing squamous cell carcinoma, treating or preventingneuroinflammatory or neurodegenerative diseases (e.g. multiplesclerosis, Parkinson's disease, Alzheimer's disease, dementia,transverse myelitis or epilepsy), treating or preventing asthma or otherbronchial and/or respiratory conditions, treating or preventingosteoporosis, bone fractures or other bone conditions, treating orpreventing eye cataracts or other eye conditions, or combinationsthereof.

In a sixth aspect, the present invention provides a method for reducinginflammation, treating or preventing inflammation associated witharthritis, promoting hair growth, treating or preventing psoriasis,increasing blood flow, treating or preventing chilblains, treating orpreventing varicose veins, treating a wound, promoting wound healing,treating or promoting healing of scar tissue, treating burns,alleviating or reducing joint pain, alleviating or reducing muscularpain, alleviating or reducing neuropathic or nerve pain, treating orpreventing sinus inflammation, treating or preventing sinus pain,treating or preventing a bacterial infection, treating or preventing afungal infection, treating or preventing eczema, treating or preventingwrinkles, treating or preventing bruises, treating or preventing jointdegeneration, treating or preventing cartilage degeneration, treating orpreventing acne, treating or preventing muscle damage, treating orpreventing motor neuron disease (MND; MND includes, for example,amyotrophic lateral sclerosis (ALS; also known as Lou Gehrig's Disease),primary lateral sclerosis (PLS), progressive muscular atrophy (PMA),progressive bulbar palsy (PBP) and pseudobulbar palsy), treating orpreventing viral conditions, treating or preventing prion diseases (e.g.bovine spongiform encephalopathy (BSE), scrapie, Creutzfeldt-Jakobdisease (CJD) or its variant (vCJD), Gerstmann-Straussler-Scheinkersyndrome, fatal familial insomnia or kuru), treating or preventing jointdamage, treating or preventing tendon damage, treating or preventingonychoschizia (split nails), treating or preventing cancer, treating orpreventing breast cancer, treating or preventing brain cancer, treatingor preventing melanoma, treating or preventing basal cell carcinoma,treating or preventing squamous cell carcinoma, treating or preventingneuroinflammatory or neurodegenerative diseases (e.g. multiplesclerosis, Parkinson's disease, Alzheimer's disease, dementia,transverse myelitis or epilepsy), treating or preventing asthma or otherbronchial and/or respiratory conditions, treating or preventingosteoporosis, bone fractures or other bone conditions, treating orpreventing eye cataracts or other eye conditions, or combinationsthereof, in a human or animal, comprising administering to the human oranimal a therapeutically effective amount of a metal ion complex or amixture of metal ion complexes obtained by the process according to thefirst aspect.

In a seventh aspect, the present invention provides a metal ion complexcomprising a ligand of Formula (I) or Formula (II)

wherein

R¹ is H and R² is H or OH, or R³ and R² together with the carbon atom towhich they are attached form a carbonyl (C═O);

R³ is H and R⁴ is H or OH, or R³ and R⁴ together with the carbon atom towhich they are attached form a carbonyl (C═O);

R⁵ is —CH(OR⁷)CH₂OR⁷ or —CH₂CO₂R⁷ and R⁶ is absent or H, or R⁵ is H andR⁶ is —CH(OR⁷)CH₂OR⁷ or —CH₂CO₂R⁷; and

each R⁷ is independently absent or H;

or a salt thereof, tautomer thereof or polymer thereof.

In an embodiment, the ligand is of Formula (I) or a salt thereof,tautomer thereof or polymer thereof.

In an embodiment, R⁵ is —CH(OR⁷)CH₂OR⁷ or —CH₂CO₂R⁷ and R⁶ is absent orH, or a salt thereof, tautomer thereof or polymer thereof.

In an embodiment, the ligand is of Formula (Ia)

wherein

R¹ is H and R² is OH, or R¹ and R² together with the carbon atom towhich they are attached form a carbonyl (C═O);

R³ is H and R⁴ is OH, or R³ and R⁴ together with the carbon atom towhich they are attached form a carbonyl (C═O);

R⁶ is absent or H; and

each R⁷ is independently absent or H;

or a salt thereof, tautomer thereof or polymer thereof.

In an embodiment, the ligand is of Formula (Ib)

wherein

R⁶ is absent or H; and

each R⁷ is independently absent or H;

or a salt thereof, tautomer thereof or polymer thereof.

In an embodiment, the ligand is of Formula (II) or a salt thereof,tautomer thereof or polymer thereof.

In an embodiment, the ligand is of Formula (IIa)

wherein

each R⁷ is independently absent or H;

or a salt thereof, tautomer thereof or polymer thereof.

In an eighth aspect, the present invention provides a metal ion complexcomprising:

-   -   a metal ion; and    -   a ligand;        wherein        the complex is a complex obtainable from the reaction of EDTA        and H_(L)OZ in the presence of copper and having an ¹H NMR in        D₂O substantially as depicted in FIG. 1, FIG. 2 or FIG. 3.

In an embodiment of the seventh or eighth aspect of the presentinvention, the metal ion complex comprises Cu²⁺, Cu³⁺ or a mixture ofCu²⁺ and Cu³⁺.

In a ninth aspect, the present invention provides a compositioncomprising a metal ion complex or a mixture of metal ion complexesaccording to the seventh or eighth aspect of the present invention, or asalt thereof, tautomer thereof or polymer thereof.

In a tenth aspect, the present invention provides a formulation fortopical administration comprising a metal ion complex or a mixture ofmetal ion complexes according to the seventh or eighth aspect of thepresent invention, or a salt thereof, tautomer thereof or polymerthereof.

In an eleventh aspect, the present invention provides the use of a metalion complex or a mixture of metal ion complexes according to the seventhor eighth aspects of the present invention, or a salt thereof, tautomerthereof or polymer thereof, in the manufacture of a medicament forreducing inflammation, treating or preventing inflammation associatedwith arthritis, promoting hair growth, treating or preventing psoriasis,increasing blood flow, treating or preventing chilblains, treating orpreventing varicose veins, treating a wound, promoting wound healing,treating or promoting healing of scar tissue, treating burns,alleviating or reducing joint pain, alleviating or reducing muscularpain, alleviating or reducing neuropathic or nerve pain, treating orpreventing sinus inflammation, treating or preventing sinus pain,treating or preventing a bacterial infection, treating or preventing afungal infection, treating or preventing eczema, treating or preventingwrinkles, treating or preventing bruises, treating or preventing jointdegeneration, treating or preventing cartilage degeneration, treating orpreventing acne, treating or preventing muscle damage, treating orpreventing motor neuron disease (MND; MND includes, for example,amyotrophic lateral sclerosis (ALS; also known as Lou Gehrig's Disease),primary lateral sclerosis (PLS), progressive muscular atrophy (PMA),progressive bulbar palsy (PBP) and pseudobulbar palsy), treating orpreventing viral conditions, treating or preventing prion diseases (e.g.bovine spongiform encephalopathy (BSE), scrapie, Creutzfeldt-Jakobdisease (CJD) or its variant (vCJD), Gerstmann-Straussler-Scheinkersyndrome, fatal familial insomnia or kuru), treating or preventing jointdamage, treating or preventing tendon damage, treating or preventingonychoschizia (split nails), treating or preventing cancer, treating orpreventing breast cancer, treating or preventing brain cancer, treatingor preventing melanoma, treating or preventing basal cell carcinoma,treating or preventing squamous cell carcinoma, treating or preventingneuroinflammatory or neurodegenerative diseases (e.g. multiplesclerosis, Parkinson's disease, Alzheimer's disease, dementia,transverse myelitis or epilepsy), treating or preventing asthma or otherbronchial and/or respiratory conditions, treating or preventingosteoporosis, bone fractures or other bone conditions, treating orpreventing eye cataracts or other eye conditions, or combinationsthereof.

In a twelfth aspect, the present invention provides a method forreducing inflammation, treating or preventing inflammation associatedwith arthritis, promoting hair growth, treating or preventing psoriasis,increasing blood flow, treating or preventing chilblains, treating orpreventing varicose veins, treating a wound, promoting wound healing,treating or promoting healing of scar tissue, treating burns,alleviating or reducing joint pain, alleviating or reducing muscularpain, alleviating or reducing neuropathic or nerve pain, treating orpreventing sinus inflammation, treating or preventing sinus pain,treating or preventing a bacterial infection, treating or preventing afungal infection, treating or preventing eczema, treating or preventingwrinkles, treating or preventing bruises, treating or preventing jointdegeneration, treating or preventing cartilage degeneration, treating orpreventing acne, treating or preventing muscle damage, treating orpreventing motor neuron disease (MND; MND includes, for example,amyotrophic lateral sclerosis (ALS; also known as Lou Gehrig's Disease),primary lateral sclerosis (PLS), progressive muscular atrophy (PMA),progressive bulbar palsy (PBP) and pseudobulbar palsy), treating orpreventing viral conditions, treating or preventing prion diseases (e.g.bovine spongiform encephalopathy (BSE), scrapie, Creutzfeldt-Jakobdisease (CJD) or its variant (vCJD), Gerstmann-Straussler-Scheinkersyndrome, fatal familial insomnia or kuru), treating or preventing jointdamage, treating or preventing tendon damage, treating or preventingonychoschizia (split nails), treating or preventing cancer, treating orpreventing breast cancer, treating or preventing brain cancer, treatingor preventing melanoma, treating or preventing basal cell carcinoma,treating or preventing squamous cell carcinoma, treating or preventingneuroinflammatory or neurodegenerative diseases (e.g. multiplesclerosis, Parkinson's disease, Alzheimer's disease, dementia,transverse myelitis or epilepsy), treating or preventing asthma or otherbronchial and/or respiratory conditions, treating or preventingosteoporosis, bone fractures or other bone conditions, treating orpreventing eye cataracts or other eye conditions, or combinationsthereof, in a human or animal, comprising administering to the human oranimal a therapeutically effective amount of a metal ion complex or amixture of metal ion complexes according to the seventh or eighth aspectof the present invention, or a salt thereof, tautomer thereof or polymerthereof.

In a further aspect, the present invention provides a metal ion complexor a mixture of metal ion complexes according to the seventh or eighthaspects of the present invention, or a salt thereof, tautomer thereof orpolymer thereof, or a metal ion complex or a mixture of metal ioncomplexes obtained by the process according to the first aspect of thepresent invention, for use in reducing inflammation, treating orpreventing inflammation associated with arthritis, promoting hairgrowth, treating or preventing psoriasis, increasing blood flow,treating or preventing chilblains, treating or preventing varicoseveins, treating a wound, promoting wound healing, treating or promotinghealing of scar tissue, treating burns, alleviating or reducing jointpain, alleviating or reducing muscular pain, alleviating or reducingneuropathic or nerve pain, treating or preventing sinus inflammation,treating or preventing sinus pain, treating or preventing a bacterialinfection, treating or preventing a fungal infection, treating orpreventing eczema, treating or preventing wrinkles, treating orpreventing bruises, treating or preventing joint degeneration, treatingor preventing cartilage degeneration, treating or preventing acne,treating or preventing muscle damage, treating or preventing motorneuron disease (MND; MND includes, for example, amyotrophic lateralsclerosis (ALS; also known as Lou Gehrig's Disease), primary lateralsclerosis (PLS), progressive muscular atrophy (PMA), progressive bulbarpalsy (PBP) and pseudobulbar palsy), treating or preventing viralconditions, treating or preventing prion diseases (e.g. bovinespongiform encephalopathy (BSE), scrapie, Creutzfeldt-Jakob disease(CJD) or its variant (vCJD), Gerstmann-Straussler-Scheinker syndrome,fatal familial insomnia or kuru), treating or preventing joint damage,treating or preventing tendon damage, treating or preventingonychoschizia (split nails), treating or preventing cancer, treating orpreventing breast cancer, treating or preventing brain cancer, treatingor preventing melanoma, treating or preventing basal cell carcinoma,treating or preventing squamous cell carcinoma, treating or preventingneuroinflammatory or neurodegenerative diseases (e.g. multiplesclerosis, Parkinson's disease, Alzheimer's disease, dementia,transverse myelitis or epilepsy), treating or preventing asthma or otherbronchial and/or respiratory conditions, treating or preventingosteoporosis, bone fractures or other bone conditions, treating orpreventing eye cataracts or other eye conditions, or combinationsthereof.

BRIEF DESCRIPTION OF THE FIGURES

Preferred embodiments of the present invention are described below, byway of example only, with reference to the accompanying drawings inwhich:

FIG. 1 is a 600 MHz ¹H NMR spectrum of “Fraction 1” in D₂O (as describedin Example 19) plotted as signal intensity vs. chemical shift (ppm).

FIG. 2 is a 600 MHz ¹H NMR spectrum of “Fraction 2” in D₂O (as describedin Example 19) plotted as signal intensity vs. chemical shift (ppm).

FIG. 3 is a 600 MHz ¹H NMR spectrum of “Fraction 6” in D₂O (as describedin Example 19) plotted as signal intensity vs. chemical shift (ppm).

FIG. 4 is a cyclic voltammogram trace showing the current (in μA, i.e.10⁻⁶ amps) vs potential (in V vs Ag/AgCl) of Sample 19.1 (as describedin Example 19).

FIG. 5 is a graphical representation showing the viability (relative tountreated cells) of MM200 cells 48 h after treatment with Sample 19.1(as described in Example 19) at different concentrations. This is asummary of the results of the melanoma cell line assay using MM200 cellsas described in Example 21.

FIG. 6 is a graphical representation showing the viability (relative tountreated cells) of MelRMu cells 48 h after treatment with Sample 19.1(as described in Example 19) at different concentrations. This is asummary of the results of the melanoma cell line assay using MelRMucells as described in Example 21.

FIG. 7a is a graphical representation showing the viability (relative tountreated cells) of MCF7 cells 48 h after treatment with Sample 19.1 (asdescribed in Example 19) at different concentrations. This is a summaryof the results of the breast cancer cell line assay using MCF7 cells asdescribed in Example 21.

FIG. 8 is a graphical representation showing the viability (relative tountreated cells) of MDA-MB-231 cells 48 h after treatment with Sample19.1 (as described in Example 19) at different concentrations. This is asummary of the results of the breast cancer cell line assay usingMDA-MB-231 cells as described in Example 21.

FIG. 9 is a graphical representation showing the viability (relative tountreated cells) of Primary Foetal Astrocyte, U87MG and T98G cells 48 hafter treatment with Sample 19.1 (as described in Example 19) atdifferent concentrations. This is a summary of the results of the humanglioblastoma cell line assay using U87MG and T98G cells (with PrimaryFoetal Astrocyte cells as control) as described in Example 21.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

In a first aspect, the present invention provides a process for thepreparation of a metal ion complex, the process comprising:

-   -   (a) contacting a metal in the form of particles having a bulk        density of between about 0.2 and about 8.0 g/cm³ with a        chelating agent in solid form; and    -   (b) while the metal is in contact with the chelating agent,        contacting the metal and chelating agent with an oxidising        agent, to form at least one metal ion complex.

In the context of the present specification, the term “metal ioncomplex” should be understood to include metal ion complexes (whethercharged or uncharged), as well as salts, protonated forms and hydratedforms thereof.

Whilst not seeking to be limited to any one proposed mechanism offormation, the process of forming the metal ion complex may involve anin situ redox reaction between the metal and the oxidising agent,whereby the metal is oxidised to one or more corresponding cations.

The metal cations thus produced may then chelate with the chelatingagent to form the metal ion complex. Suitable combinations of oxidisingagent and metal can be selected based on the reduction potential of therespective reagents. Reduction potentials are known to persons skilledin the art and are reported, for example, in the CRC Handbook ofChemistry and Physics, Weast, R., Ed. 55^(th) Edition, 1974-1975.

By way of illustration, the following reaction may be envisaged, wherethe oxidising agent is hydrogen peroxide and M is a suitable metal:

M_((s))+H₂O₂+2H⁺

M²⁺+2H₂O

The resultant M²⁺ ions are then able to form a metal ion complex with aligand (e.g. a chelating agent).

The inventors have found that if metal in the form of particles having abulk density of between about 0.2 and about 8.0 g/cm³ is contacted withthe chelating agent in solid form before the commencement of theoxidation reaction, and the reaction allowed to proceed to completion,metal ion complexes can be prepared that have different properties tometal ion complexes prepared when the chelating agent is in solutionwhen the chelating agent is contacted with the metal.

Bulk density is a property of powders, granules, and other “divided”solids or particulate matter. It is the mass of the particles of thematerial divided by the total volume they occupy. The total volumeincludes particle volume, inter-particle void volume, and internal porevolume. The bulk density of the metal particles may be determined by,for example, adding 100 g of the metal particles to a 200 ml measuringcylinder (graduated cylinder). The initial volume measured in themeasuring cylinder is the “freshly settled” volume. The measuringcylinder is then picked up and dropped 3 times from a height of 2 cmonto a solid surface. The volume measured in the measuring cylinder atthis stage is the “tapped” volume. The bulk density is thus determinedby the formula:

${{bulk}\mspace{14mu} {density}\mspace{14mu} ( {{in}\mspace{14mu} g\text{/}{mL}\mspace{14mu} {or}\mspace{14mu} g\text{/}{cm}^{3}} )} = \frac{100}{{measured}\mspace{14mu} {volume}\mspace{14mu} ( {{in}\mspace{14mu} {mL}\mspace{14mu} {or}\mspace{14mu} {cm}^{3}} )}$

As used herein, all references to the bulk density of metal particlesrefer to the “tapped” bulk density, as determined by the proceduredescribed above unless expressly stated otherwise. In the process of thepresent invention, a metal in the form of particles having a bulkdensity of between about 0.2 and about 8.0 g/cm³ is used. In variousembodiments, the metal has a bulk density of between about 0.3 and about4.0 g/cm³, for example, between about 0.5 and about 3.0 g/cm³, betweenabout 0.5 and about 2.5 g/cm³, between about 0.7 and about 2.0 g/cm,between about 0.8 and about 1.7 g/cm³ or between about 0.9 and about 1.5g/cm³.

In the process of the present invention, the metal may be any metalcapable of forming a metal ion complex with the chelating agent. Themetal used in the process is typically in its elemental state (i.e. anoxidation state of 0). The metal may, however, have an oxide (or other)layer on the metal in its elemental state. In an alternative embodiment,the metal is a metal salt which is oxidised to a higher oxidation stateduring the process to form the metal ion complex. For example, a Cu(I)salt (i.e. comprising Cu⁺) may be oxidised to the Cu (II) salt (i.e.comprising Cu²⁺) or to the Cu (III) salt (i.e. comprising Cu³⁺), whichthen goes on to form the metal ion complex.

The metal is in the form of particles. The metal may, for example, be inthe form of metal turnings, wire, ribbon, granules, powder, a solid barhaving any shape, or any other suitable particulate form.

In some embodiments, the metal particles may have an oxide (or other)layer, or develop an oxide (or other) layer prior to being used in theprocess. In these embodiments, the term “contacting the metal” is takento apply to contacting the bulk metal particles (i.e. the metalincluding any oxide (or other) layer) with the appropriate agent. Thismay occur by contacting the oxide (or other) layer exclusively, orcontacting the oxide (or other) layer and the elemental metalsimultaneously (both the elemental metal and layer at the same time).The process of the invention embraces both of these alternatives, solong as the metal is able to react with the other agents. In someembodiments the metal may be pre-treated prior to the process in orderto remove or reduce an oxide (or other) layer on the metal.

The chelating agent may be any compound capable of forming a metal ioncomplex with a cation of the metal. Metal ion complexes (also known ascoordination complexes) formed by the process of the present inventioncomprise a metal cation having two or more coordinate bonds to thechelating agent. The chelating agent is thus a ligand having more thanone site (e.g. functional group) capable of forming a coordination bondwith a metal cation. The chelating agent may be described as being amultidentate (also known as polydentate) ligand.

Chelation between the metal and the chelating agent involves theformation or presence of two or more separate coordinate bonds between amultidentate (i.e. multiple bonded) ligand and a central atom (i.e.metal cation).

The chelating agent (i.e. multidentate ligand) may be charged oruncharged, so long as it is capable of forming a coordinate bond with acation of the metal to form a metal ion complex. In some embodiments,the chelating agent (ligand) is an organic compound.

Chelating agents are also sometimes called chelants, chelators orsequestering agents.

In coordination chemistry, the bonding between a metal cation and aligand generally involves formal donation of one or more of the ligand'selectron pairs. The nature of metal-ligand bonding can range fromcovalent to ionic. Furthermore, the metal-ligand bond order can rangefrom one to three. Ligands are sometimes viewed as Lewis bases, althoughrare cases are known to involve Lewis acidic ligands.

As used herein, the term “chelating agent” includes within its scopecompounds capable of forming a multidentate ligand in situ under theconditions of the process of the present invention (i.e. a “chelatingagent precursor”). In other words, the chelating agent contacted withthe metal in the process of the present invention may be a compound thatis not necessarily capable of acting as a multidentate ligand, but istransformed to a substance capable of acting as a multidentate ligandunder the conditions experienced during the process of the invention.

The chelating agent is in solid form in step (a) of the process of thepresent invention. In this context, “solid form” refers to the chelatingagent being a solid. Typically the chelating agent is in the form of agranulated or powdered solid. At least a portion of the chelating agentis in solid form when contacting the metal and when the metal andchelating agent is first contacted with the oxidising agent. Withoutwishing to be bound by theory, the inventors believe that the metalbeing in contact with the chelating agent in solid form when the metaland the chelating agent is contacted with the oxidising agent leads to aconcentration gradient being established between the metal and thechelating agent in the resultant reaction mixture. Without wishing to bebound by theory, the inventors believe that, as a result of thisconcentration gradient, the process of the present invention can resultin the formation of different metal ion complexes to those formed whenthe chelating agent is in solution prior to the chelating agent beingcontacted with the metal.

In an embodiment, the process of the invention forms a single species ofmetal ion complex. In other embodiments, the process of the inventionforms multiple species of metal ion complexes (i.e. a mixture of metalion complexes). These multiple species may be produced simultaneously orsequentially, and may be kept together as a mixture, or separated in asubsequent step.

The process of the present invention comprises contacting a metal with achelating agent. In an embodiment, the metal is added to the chelatingagent. In another embodiment, the chelating agent is added to the metal.In either of these embodiments, the end result is that the metal andchelating agent are in contact with each other. In other words, themetal and chelating agent are in intimate physical contact. In someembodiments, the metal and chelating agent are combined and mixed toform a mixture in which the metal and chelating agent are in contactwith each other.

The process of the present invention comprises contacting the metal andchelating agent (while the metal is in contact with the chelating agent)with an oxidising agent. In an embodiment, the metal in contact with thechelating agent is added to the oxidising agent. In another embodiment,the oxidising agent is added to the metal and chelating agent (while themetal is in contact with the chelating agent).

In an embodiment, the oxidising agent is added to the metal/chelatingagent at one time. In another embodiment, the metal/chelating agent isadded to the oxidising agent at one time. Alternatively, in otherembodiments, the oxidising agent is added to the metal/chelating agentin a portionwise fashion over a period of time. For example, theoxidising agent may be added portionwise over about 5 seconds to about 1hour or 2 to 3 weeks. In other embodiments, the metal/chelating agent isadded to the oxidising agent in a portionwise fashion over a period oftime as above. In further alternative embodiments, the addition of theoxidising agent to the metal/chelating agent, or the metal/chelatingagent to the oxidising agent, is performed in a continuous fashion overa period of time. For example, addition can be made by a syringe pump orsolids addition funnel or other apparatus known to those skilled in theart. Those skilled in the art would be able to gauge an appropriate rateof addition taking into consideration factors such as concentration,temperature, reagents, etc.

In an embodiment, the process of the invention is carried out as a “onepot” process. In some embodiments, the process is carried out in astepwise fashion, including stepwise addition of the oxidising agentand/or metal/chelating agent to the other.

In an embodiment, the metal/chelating agent is wetted with a solventprior to contacting the oxidising agent. In some embodiments themetal/chelating agent is wetted with water prior to contacting theoxidising agent. In this regard, “wetted” refers to the application ofan amount of solvent or water to the metal/chelating agent. Thistypically involves applying an amount sufficient to cover themetal/chelating agent.

The process of the present invention typically comprises the furtherstep of allowing the combination of reagents resulting from the contactof the metal and chelating agent with the oxidising agent to react untilcompletion. In other words, the oxidation reaction(s) initiated by theoxidising agent is(are) allowed to go to completion. Accordingly, in anembodiment, the present invention provides a process for the preparationof a metal ion complex, the process comprising:

-   -   (a) contacting a metal in the form of particles having a bulk        density of between about 0.2 and about 8.0 g/cm³ (e.g. between        about 0.3 and about 4.0 g/cm³) with a chelating agent in solid        form;    -   (b) while the metal is in contact with the chelating agent,        contacting the metal and chelating agent with an oxidising        agent; and    -   (c) allowing the resulting combination to react until        completion.

In the context of the process of the present invention, there areseveral characteristics to indicate to a person skilled in the art thatthe reaction has gone to completion.

In an embodiment, the completion of the reaction may be indicated by an“exothermic explosion”. The “exothermic explosion” may be considered aperiod towards the end of the reaction where the reaction enters anexothermic phase. For example, the exothermic reaction may lead to athermal runaway or a runaway reaction whereby the heat generated fromthe exothermic nature of the reaction(s) increases the rate of thereaction in a positive feedback, leading to a period of highlyexothermic reaction. In embodiments that contain solvents, particularlylow-boiling solvents, or any other low-boiling reagent or component,these may be quickly converted to their gaseous states and have theappearance of an explosion. Other indicators that the reaction isreaching completion may include the release of water vapour, CO₂, ozone,oxygen and/or other gaseous products. In some embodiments, the reactionmixture appears to “boil” with bubbles of gaseous products. For example,and without wishing to be constrained by theory, in the case of anembodiment using Cu as the metal, disodium EDTA as the chelating agentand HO, as the oxidising agent, there is believed to be adecarboxylation event as the reaction nears completion, which affordsbubbles of CO₂.

In other embodiments, the completion of the reaction may be indicated bya change in the colour of the reaction. In one embodiment that employscopper as the metal, the reaction mixture changes colour from blue to agreenish blue colour, indicating the completion of the reaction (e.g.from Pantone® 2386 C to Pantone® 306 UP).

In other embodiments, the completion of the reaction may be indicated bythe formation of specific metal ion complexes as determined bychromatographic techniques, such as, for example, HPLC.

Allowing the combination to react until completion comprises allowingthe combination to react for a sufficient period of time for thereaction to proceed until completion. In some embodiments, allowing theresulting combination to react until completion involves allowing thecombination to react for extended periods, for example, the combinationmay be left for 2 to 5 weeks in order to go to completion. In someembodiments, the combination will react for 1 day, 2 to 7 days, 1 to 2weeks or 1 to 3 weeks before entering an exothermic phase. Inembodiments where the reaction mixture is heated at the outset, it ismore likely that the reaction will go to completion within a shorterperiod of time. For example, the reaction may go to completion in about5 to 15 mins, about 15 to 30 mins about 30 mins to 1 hour or about 1 to2 hours, when the reaction components are heated.

In some embodiments, the combination will react until all of theoxidising agent is consumed. In other embodiments, the combination willreact until all of the chelating agent is consumed. In other embodimentsstill, the combination will react until all of the metal is consumed. Insome embodiments, the process further comprises a step of removingunreacted metal and/or unreacted chelating agent and/or unreactedoxidising agent.

In an embodiment, mixtures of different metals are used. In anembodiment, the metal used in the process is an alloy comprising two ormore metals, with at least one metal being capable of forming a metalion complex. In another embodiment, the alloy comprises two or moremetals with more than one metal being capable of forming a metal ioncomplex.

In an embodiment, the metal and chelating agent are above ambienttemperature (for example at above about 30° C., above about 40° C.,above about 50° C., above about 60° C., above about 70° C., above about75° C., above about 80° C. or at about 85° C.; e.g. in the range ofabout 30° C. to about 100° C., about 30° C. to about 90° C., about 30°C. to about 80° C., about 40° C. to about 90° C., about 60° C. to about90° C. or about 80° C. to 90° C.) when the oxidising agent is contactedwith the metal and chelating agent. In some embodiments, the metal andchelating agent are heated before the oxidising agent is contacted withthe metal and chelating agent.

In an embodiment, the oxidising agent is above ambient temperature whenit is contacted with the metal and chelating agent. In an embodiment theoxidising agent is hot when contacting the metal and chelating agent.For example, the oxidising agent may be at above about 30° C., aboveabout 40° C., above about 50° C., above about 60° C., above about 70°C., above about 75° C., above about 80° C. or at about 85° C.; e.g. inthe range of about 30° C. to about 100° C., about 30° C. to about 90°C., about 30° C. to about 80° C., about 40° C. to about 90° C., about60° C. to about 90° C. or about 80° C. to 90° C.

In some embodiments, both the metal/chelating agent and the oxidisingagent are above ambient temperature.

In some embodiments, both the metal/chelating agent and the oxidisingagent are above ambient temperature when contacting each other.

In an embodiment, the metal and chelating agent are heated when theoxidising agent is contacted with the metal and chelating agent. In anembodiment, heat is applied to the metal before it contacts thechelating agent, thereby elevating the temperature of themetal/chelating agent above ambient temperature. In an alternativeembodiment, heat is applied to the chelating agent before it contactsthe metal, thereby elevating the temperature of the metal/chelatingagent above ambient temperature. In another embodiment, heat is appliedto the metal/chelating agent while the metal is in contact with thechelating agent, and before the oxidising agent is contacted with themetal and chelating agent, in order to bring it to above an ambienttemperature. In another embodiment, heat is applied to themetal/chelating agent whilst the oxidising agent contacts themetal/chelating agent.

In an embodiment, heat is applied during step (b) to raise thetemperature above ambient. In an embodiment, the heat is applied duringstep (c) to raise the temperature above ambient.

In some embodiments heat is applied continuously, whilst in otherembodiments the heat is applied at time intervals that may be regular orirregular. Heat may be applied, for example, by means of a heatingmantle, heating jacket, hotplate, microwave or any other means, or anycombination thereof, in order to raise the temperature above ambienttemperature.

Modification of the temperature may be used to control the rate ofreaction. Accordingly, the process may optionally include one or moreheating or cooling steps at any stage. For example, heat may be appliedto the reaction in order to encourage the reaction to go to completionin a shorter period of time.

In an embodiment, the ratio of metal:chelating agent in moles is withinthe range of about 1:5 to about 100:1. In various embodiments, the ratioof metal:chelating agent is within the range of about 1:1 to about 50:1,about 1:1 to about 40:1, about 1:1 to about 10:1, about 2:1 to about30:1, about 3:1 to about 20:1, about 4:1 to about 20:1, or about 4:1 toabout 10:1. In some embodiments the ratio is about 5:1. In someembodiments, an excess of metal is used.

In an embodiment, the ratio of chelating agent:oxidising agent in molesis within the range of about 2:1 to about 1:100. In various embodiments,the ratio of chelating agent:oxidising agent is within the range ofabout 1:1 to about 1:50, about 1:1 to about 1:20, about 1:2 to about1:20, about 1:2 to about 1:10 or about 1:3 to about 1:5. In someembodiments the ratio is about 1:3.3.

In an embodiment, the metal ion complex prepared by the process of theinvention is water soluble.

In an embodiment, the amount of metal added is sufficient to consumesubstantially all of the oxidising agent. In another embodiment, theamount of chelating agent added is sufficient to complex substantiallyall of the metal ions.

In some embodiments, adding an excess of the chelating agent may slowthe reaction down.

In an embodiment, about 50 to about 500 grams of metal is used. Invarious other embodiments, about 100 to about 1000 grams, about 0.5 kgto about 5 kg, about 1 kg to about 10 kg, about 5 kg to about 50 kg,about 10 kg to about 100 kg, about 50 kg to about 250 kg of metal isused.

In one embodiment the resultant metal ion complex is a salt, such as analkali earth or alkali metal salt. For example, the metal ion complexmay be a Na⁺, K⁺, Li⁺, Mg²⁺, or Ca²⁺ salt.

In an embodiment, the metal to be used in the process of the inventionis a transition metal. In another embodiment the metal is an alkalineearth metal. In an embodiment the metal is selected from the groupconsisting of Cu, Mn, Fe, Co, Ni, Zn, Cr, Al, Cd, Ag and Se and mixturesthereof.

In one embodiment the metal is copper.

In one embodiment the metal is copper which is oxidised to Cu³⁺, Cu²⁺ orCu⁺ ions, or a mixture thereof. In another embodiment the metal is ironwhich is oxidised to Fe²⁺ or Fe³⁺ ions, or a mixture thereof.

In a further embodiment the metal is zinc which is oxidised to Zn²⁺ions. In another embodiment the metal is Ni which is oxidised to Ni²⁺ions. In a further embodiment the metal is cobalt which is oxidised toCo²⁺ or Co³⁺ ions, or a mixture thereof. In another embodiment the metalis silver which is oxidised to Ag⁺ ions.

Without wishing to be bound by theory, it is believed that one factor inthe observed biological activity of copper complexes prepared by theprocess of the present invention as described in Examples 20 to 22 maybe due to the formation of relatively stable complexes of Cu³⁺.

In an embodiment, the chelating agent is a multidentate ligand capableof forming a stable metal ion complex. In an embodiment, the chelatingagent is bidentate. In an embodiment, the chelating agent is tridentate.In an embodiment, the chelating agent is tetradentate. In an embodiment,the chelating agent is pentadentate. In an embodiment, the chelatingagent is hexadentate.

In an embodiment, the chelating agent is neutral. In an embodiment, thechelating agent is positively charged. In an embodiment, the chelatingagent is negatively charged. In an embodiment, the chelating agent iszwitterionic. In an embodiment, the chelating agent is used as itscorresponding hydrate. In embodiments that include charged chelatingagents, the chelating agent may be used as any suitable salt (i.e. acharged chelating agent with any suitable counterion).

In an embodiment, the chelating agent comprises a nitrogen and/or anoxygen donor. In this regard, the nitrogen and/or oxygen atom is part ofa functional group on the chelating agent. The nitrogen and/or oxygenatom is able to donate electrons to the metal centre to thus form acoordinate bond and thus the coordinate complex (i.e. metal ioncomplex). In some embodiments, the donor on the chelating agent is anitrogen. In some embodiments, the donor on the chelating agent is anoxygen. In some embodiments, the chelating agent comprises both anitrogen and an oxygen donor. In some of such embodiments, not all ofthe nitrogen and/or oxygen donors form a coordinate bond with the metalcentre. In some embodiments, the chelating agent comprises a sulfurdonor.

In an embodiment the chelating agent comprises at least one carboxylicacid (or carboxylate) group. In one embodiment the chelating agentcomprises at least one amino group, wherein the amino group may be aprimary, secondary or tertiary amino group. In one embodiment thechelating agent may comprise at least one, e.g. 1, 2, 3, 4, 5 or 6,imidodiacetic acid [—N(CH₂CO₂H)₂] groups, wherein one or both of themethylene (—CH₂) hydrogen atoms may be replaced with anothersubstituent, such as a C₁-C₄ alkyl group.

In an embodiment, the chelating agent is selected from the groupconsisting of ethylenediaminetetraacetic acid (EDTA),diethylenetriaminepentaacetic acid (DTPA),hydroxyethylenediaminetetraacetic acid (HEDTA),ethylenediaminedisuccinic acid (EDDS), salicylic acid, acetyl salicylicacid, amino acids, peptides, and salts and hydrates thereof. Thestructures of EDTA, DTPA, HEDTA and EDDS are shown below:

EDTA is a hexadentate chelating ligand with 6 possible coordinationsites. The pKa of the carboxylic acid residues of EDTA are 1.70, 2.60,6.30 and 10.60, respectively.

Neutral EDTA can exist as a zwitterion with one or two protons locatedon one or two of the nitrogen atoms.

EDTA, DTPA, and HEDTA have a strong affinity for a wide range of metalions including Cu²⁺, Ag⁺, Ca²⁺, Co³⁺, Fe³⁺, Fe²⁺, Mg²⁺, Ni²⁺ and Zn²⁺.

In one embodiment the chelating agent comprises amino acids such asglycine, histidine, lysine, arginine, cysteine, methionine, and peptidescomprising those amines, and salts thereof. The peptide may comprisetwo, three, four or five amino acids. The respective amino acids may bethe same or different. In one embodiment, the peptide is a dipeptide ora tripeptide.

In one embodiment the chelating agent is EDTA or a salt thereof. In anembodiment, the salt of EDTA comprises one or more of sodium ions,potassium ions, lithium ions, calcium ions, magnesium ions, or mixturesthereof. In one embodiment, the chelating agent is EDTA disodium salt.In another embodiment the chelating agent is DTPA or a salt thereof, ora sodium salt thereof.

At least a portion of the chelating agent is in solid form when themetal and chelating agent are first contacted with the oxidising agentin step (b). In an embodiment, at least a portion of the chelating agentremains in solid form during step (c).

In an embodiment, the oxidising agent is selected from the groupconsisting of peroxides, peracids, ozone and oxidising salts. In anembodiment, the oxidising agent is an N-oxide such as TEMPO.

In an embodiment, the oxidising agent is in solution when contacting themetal and chelating agent. The solvent used to make the solution may beany suitable solvent. In order to be suitable, the oxidising agentshould not react with (or react to any appreciable amount with) thesolvent. In addition, the oxidising agent should maintain its ability toact as an oxidising agent while in solution. In an embodiment, theoxidising agent is in an aqueous solution. In an embodiment, the aqueoussolution comprises a cosolvent.

In an embodiment, the oxidising agent is hydrogen peroxide. In anembodiment, the hydrogen peroxide is in an aqueous solution.

In an embodiment, the aqueous solution of hydrogen peroxide compriseshydrogen peroxide in an amount within the range of about 0.01 wt. % toabout 100 wt. %. For example, in various embodiments, the aqueoussolution of hydrogen peroxide is within the range of about 0.01 wt. % toabout 70 wt. %, about 0.5 wt. % to about 60 wt. %, about 1 wt. % toabout 60 wt. %, about 1 wt. % to about 15 wt. %, about 15 wt. % to about30 wt. %, about 20 wt. % to about 30 wt. % about 30 wt. % to about 40wt. %, or about 45 wt. % to about 55 wt. %. In an embodiment, theaqueous solution of hydrogen peroxide is about 50 wt. %.

In an embodiment, the metal is Cu, the chelating agent isethylenediaminetetraacetic acid (EDTA) disodium salt and the oxidisingagent is hydrogen peroxide. In a further embodiment, the hydrogenperoxide is in an aqueous solution, the ratio ofCu:ethylenediaminetetraacetic acid (EDTA) disodium salt in moles isabout 1:1 to about 50:1 and the ratio of Cu:hydrogen peroxide in molesis about 1:1 to about 1:20.

In another embodiment, Cu in contact with ethylenediaminetetraaceticacid (EDTA) disodium salt is maintained at above about 75° C. when theCu and EDTA are contacted with the hydrogen peroxide, the hydrogenperoxide also being at above about 75° C. In this embodiment, the Cu andEDTA disodium salt may be mixed by any suitable mixing techniques knownto those skilled in the art. For example, mixing may be performed bymechanical or magnetic stirring, sonication, shaking, swirling, folding,whipping, inverting the reaction vessel etc.

In an embodiment, the process further comprises a step of removingunreacted metal and/or unreacted chelating agent and/or unreactedoxidising agent.

Metal ion complexes prepared according to the present invention may bemore stable at a particular pH. For example, metal ion complexesaccording to the present invention may be stable at a pH in the rangefrom about 4 to about 12, preferably in the range from about 4 to about9. Accordingly, the metal ion complex may be combined with a buffer or apH adjusting agent to provide a stable composition comprising the metalion complex.

In some embodiments, the metal ion complex comprises the chelate (i.e.ligand) and metal ion in a 1:1 stoichiometric ratio or 1:2stoichiometric ratio (i.e. 1:1 or 1:2 chelate:metal ion). In otherembodiments, the metal ion complex comprises the chelate and metal ionin a chelate:metal ion stoichiometric ratio of between 1:3 and 1:8, forexample 1:3, 1:4, 1:5, 1:6, 1:7 or 1:8. Suitable counterions may includepharmaceutically acceptable ions such as sodium ions, potassium ions,calcium ions, magnesium ions, etc.

In some embodiments, the metal ion complex comprises multiple metalcations (i.e. one or more ligands are coordinated to two or more metalcations). In some embodiments, oligomers of metal ion complexes areformed.

In some embodiments, the process comprises an additional ion exchangestep in order to exchange counterions. Techniques for performing such astep, such as ion exchange chromatography and recrystallization, areknown to those skilled in the art. Thus, for example, a salt orprotonated form of a metal ion complex (or mixture thereof) may beconverted into another salt of choice.

The process according to the invention may further include an additionalstep of isolating the metal ion complex from solution. Suitable methodsof isolation include solvent evaporation, recrystallisation, solventextraction, and other general methods known to those skilled in the art.In one embodiment, the mixture resulting from the process is evaporatedor lyophilized to obtain a solid comprising the metal ion complex. Themetal ion complex may be purified, e.g. by chromatographic techniques orby recrystallization from a suitable solvent.

The inventors have found that novel metal ion complexes comprising aligand of Formula (I) or Formula (II) as defined below can be formed bythe process according to the first aspect of the present invention.

Accordingly, in an aspect, the present invention provides a metal ioncomplex comprising a ligand of Formula (I) or Formula (II)

wherein

R¹ is H and R² is H or OH, or R¹ and R² together with the carbon atom towhich they are attached form a carbonyl (C═O);

R³ is H and R⁴ is H or OH, or R³ and R⁴ together with the carbon atom towhich they are attached form a carbonyl (C═O);

R⁵ is —CH(OR⁷) CH₂OR⁷ or —CH₂CO₂R⁷ and R⁶ is absent or H, or R⁵ is H andR⁶ is —CH(OR⁷)CH₂OR⁷ or —CH₂CO₂R⁷; and

each R⁷ is independently absent or H;

or a salt thereof, tautomer thereof or polymer thereof.

Such metal ion complexes can be prepared by the process according to thepresent invention. For example, the inventors have found that when theprocess of the present invention is carried out using the metal copper,the chelating agent EDTA and the oxidising agent hydrogen peroxide, andthe reaction is allowed to proceed to completion, the metal ioncomplexes prepared by the process include copper ion complexescomprising the ligand of Formula (III) or Formula (IV) described below(where M in Formula (III) or Formula (IV) is Cu²⁺ or Cu³⁺). Metal ioncomplexes comprising a ligand of Formula (I) or Formula (II) can also beformed by other methods. For example, the ligand may be synthesised bymethods known in the art, and reacted with (or coordinated to) a metal(or metal cation) to form the metal ion complex.

In the ligands of Formula (I) and Formula (II), the substituents R andR⁷ may, in some instances, represent H or may be absent. As a personskilled in the art will appreciate, if R⁶ or R⁷ are absent, the valencyof the heteroatom to which the R⁶ or R⁷ is attached (i.e. the N and/or Oatom) will be assumed to be satisfied by a lone pair of electrons(resulting in a formal negative charge on the heteroatom).

In such instances, the lone pair of electrons/negative charge may, forexample, form a salt with another cation (such as Na⁺, K⁺, Ca²⁺ etc.) ormay, for example, coordinate (i.e. bond) with a metal cation to form acoordination bond (sometimes known as a dative bond) and thus contributeto forming the coordination complex (i.e. metal ion complex). In otherwords, the negative charge of the —O⁻, —N⁻— or —CO₂ ⁻ group (provided bythe lone pair of electrons) is available for bonding with a proton (H⁺),cation (such as Na⁺, K⁺, Ca²⁺ etc.) or the metal ion of the metal ioncomplex.

For example, in the ligands of Formula (I) or Formula (II) —CO₂R⁷ mayrepresent —CO₂H (i.e. a carboxylic acid) when R⁷ is H, or —CO₂ ⁻ (i.e. acarboxylate anion) when R⁷ is absent. Depending on the conditions (pHfor example), a carboxylic acid (i.e. —CO₂H) may deprotonate to affordthe corresponding carboxylate anion (i.e. —CO₂ ⁻). Also depending on theconditions, the carboxylate anion may form a salt, for example —CO₂Na(sometimes depicted as —CO₂ ⁻Na⁺), or may coordinate with the metal ionof the metal ion complex, forming a coordinate bond. This may bedepicted, for example, as —CO₂ML_(n), where M represents the metal ionof the metal ion complex and L_(n) represents n ligands or ligatinggroups where n is an integer. Examples of ligating groups may includefunctional groups (such as —O⁻, —OH, —N⁻—, —NH—, —CO₂H, —CO₂ ⁻, etc.) ona ligand.

The nature of the coordinate bond, in terms of covalent or ioniccharacter, between a ligand and a metal ion will depend on a number offactors. As a person skilled in the art will appreciate, a bond betweena ligand and a metal ion may be ionic, covalent or somewhere in between.The degree of the partial ionic character (or partial covalentcharacter) of a bond may depend on, for example, the electronegativityof the metal ion and/or the electronegativity of the ligating groupinvolved. For example, when the difference in electronegativity betweenthe metal ion and the ligating group is greater, the bond will have amore ionic character. Similarly, when the difference inelectronegativity between the metal ion and the ligating group issmaller, the bond will have a more covalent character. The metal ioncomplexes of the seventh aspect of the present invention comprise aligand of Formula (I) or Formula (II), as described above, bound to ametal ion by 2 or more bonds that may be ionic, covalent, partiallyionic, partially covalent or may have any degree of partial ioniccharacter or partial covalent character.

The metal ion complexes of the seventh aspect of the present inventionmay form tautomers (i.e. may exist in multiple tautomeric forms). Aperson skilled in the art will understand that tautomers are structuralisomers that exist as a rapidly-interconverting mixture in equilibrium.The ratio of the tautomers depends on various factors, for example,temperature, solvent and pH. Most commonly, tautomers differ by theposition of a proton. In other words, a deprotonation/protonationsequence occurs to relocate a proton. When the ligands of Formula (I) orFormula (II) have a tautomer, each tautomer is embraced in the relevantFormula. For example, Formula (I) embraces both tautomers depicted inthe below partial structures:

As a further example of tautomeric forms, Formula (I) embraces bothtautomers depicted in the below partial structures:

The ligands of Formula (I) or Formula (II) may exist in stereoisomericforms (e.g. enantiomers or diastereomers depending on the number ofchiral centers). Mixtures of diastereomers and mixtures of enantiomers(e.g racemates) can be separated into the stereoisomerically uniformconstituents by methods known to persons skilled in the art. Unlessotherwise stated, it is intended that any reference herein to a compoundor ligand that is capable of existing in stereoisomeric forms (e.g. ametal ion complex comprising a ligand of Formula (I) or Formula (II)) isintended to encompass all possible stereoisomers and mixtures ofstereoisomers of the compound or ligand (e.g. is intended to encompassall possible enantiomers, mixtures of enantiomers, diastereomers ormixtures of diastereomers).

In some embodiments, the metal ion complex comprising a ligand ofFormula (I) or Formula (II) comprises a metal ion selected from an ionof Cu, Zn, Mn, Fe, Co, Ni, Cr, Al, Cd, Ag, Au and Se (e.g. Cu²⁺, Cu³⁺,Zn²⁺, Fe²⁺, Fe³⁺, Co²⁺, Co³⁺, Ni²⁺ or Ag⁺).

A metal ion complex comprising a ligand of Formula (I) or Formula (II)may form a polymer. As used herein, the terms “polymer” and “polymers”are used in the broadest sense and include an oligomer, oligomers,oligomeric material and the like, unless the context clearly requiresotherwise. That is, the terms “polymer” and “polymers” as used hereinare intended to include dimers, trimers, tetramers etc. (i.e. comprising2, 3, 4 etc. monomeric/repeating units or entities). For example, theterm “polymer” may be used herein to refer to 2, 3, 4, 20, 50, 100,1000, 10000, 100000 etc. monomeric/repeating units or entities.

In an embodiment, a polymer of the metal ion complex comprising a ligandof Formula (I) or Formula (II) may be provided by the polymerisation ofa ligand of Formula (I) and/or Formula (II). A person skilled in the artwill understand that the ligands of Formula (I) and Formula (II) eachhave functional groups capable of forming covalent bonds (e.g. esters,amides, ethers etc.). Accordingly, polymers may, in various embodiments,be formed by the polymerisation of the functional groups of the ligandsof Formula (I) and/or Formula (II) leading to polyesters, polyamides,polyethers etc. For example, a carboxylic acid group residing on theligand of Formula (I) may form an ester with an alcohol group (—OH) oneither a ligand of Formula (I) or Formula (II) to form an ester bond.

In another embodiment, the polymer of the metal ion complex comprising aligand of Formula (I) or Formula (II) is a coordination polymer. Acoordination polymer may be described as a structure containing metalcations linked by organic ligands. A coordination polymer may also bedescribed as a coordination compound with repeating coordinationentities extending in 1, 2 or 3 dimensions, or as a polymer whosemonomeric/repeat units are coordination complexes. Coordination polymersmay extend along a single dimension (and may include cross-links betweentwo or more individual chains), or may extend in 2 or 3 dimensions. Insome embodiments, the coordination polymer comprises a metal cationbound to more than 1 (e.g. 2, 3, 4, 5 or 6) ligands of Formula (I)and/or Formula (II). In other embodiments, the coordination polymercomprises ligand of Formula (I) and/or Formula (II) bound to more than 1(e.g. 2, 3, 4, 5 or 6) metal cations.

Polymers of metal complexes comprising a ligand of Formula (I) and/orFormula (II) may form during the formation of the complex, or may formduring the formation of the ligands. In other words, the metal complexesmay polymerise during their formation, or the ligands may be polymerisedprior to formation of the metal ion complex.

Polymers may be formed solely from metal ion complexes comprising aligand of Formula (I), or may be formed solely from metal ion complexescomprising a ligand of Formula (II) (i.e. homopolymers). Alternatively,polymers may also be formed from a mixture of metal ion complexescomprising a ligand of Formula (I) and metal ion complexes comprising aligand of Formula (II) (i.e. copolymers). The copolymers may, forexample, be random copolymers, alternating copolymers or blockcopolymers.

The polymers may, for example, comprise between 2 and 20000monomeric/repeating units/entities, for example, between about 2 andabout 15000 monomeric units, between about 5 and about 10000 monomericunits, between about 10 and about 5000 monomeric units, between about 20and about 2000 monomeric units, between about 100 and 1000 monomericunits or between about 500 and 5000 monomeric units.

The metal ion complexes of the eighth aspect of the present inventionmay also form polymers, as discussed above.

In the metal ion complexes of the seventh aspect of the presentinvention, the ligand may be coordinated to the metal ion of the metalion complex by between 2 and 6 ligating groups (e.g. donor atoms). Forexample, the ligand may be coordinated to the metal ion of the metal ioncomplex by between 2 and 4 ligating groups (e.g. donor atoms) or bybetween 4 and 6 ligating groups (e.g. donor atoms). For example, theligand may be attached to the metal ion of the metal ion complex by 2,3, 4, 5 or 6 bonds between the metal ion and the ligand of Formula (I)or Formula (II). In other words, bonds occur between 2, 3, 4, 5 or 6ligating groups (e.g. donor atoms) of the ligand and the metal ion ofthe metal ion complex. In an embodiment, the metal ion complex comprisesa ligand of Formula (I) or Formula (II) and a metal ion, wherein theligand is coordinated to the metal ion by 4 coordinate bonds. In anotherembodiment, the metal ion complex comprises a ligand of Formula (I) orFormula (II) and a metal ion, wherein the ligand is coordinated to themetal ion by 6 coordinate bonds.

It is likely that the ligating groups of the ligands of Formula (I) orFormula (II) are the donor atoms N and/or O. These donor atoms may bepresent in various functional groups and may be in their charged oruncharged states. For example, the metal ion may bond to the donor atomsN and/or O, which are present in the ligands of Formula (I) or Formula(II) in the functional groups —OH, —O⁻, —NH—, —N⁻—, CO₂H, CO₂ ⁻, ═N— or═O.

For example, in some embodiments, the metal ion complex may comprise aligand attached to a metal ion by 4 bonds, as represented by Formula(III) or Formula (IV) as depicted below (where M is a metal ion):

As a person skilled in the art will appreciate, the structures depictedin Formula (III) and Formula (IV) are a generalisation only, as variousprotonation/deprotonation steps will occur to give rise to tautomers ofthe structures of Formulas (III) and (IV). For example, the imidazole ofFormula (IV) may deprotonate and tautomerise to give a structure ofFormula (IVa):

As a person skilled in the art will also appreciate, the ligands mayalso bind to the metal ion by further coordination bonds. For example,one of the pendant carboxylic acid groups of Formula (III) may also bindto the metal centre to give the structure depicted in Formula (IIIa)below:

Further coordination may afford the structure depicted in Formula(IIIb):

Without wishing to be bound by theory, it may also be possible for aligand to coordinate to the metal ion via a contiguous series of 2 ormore atoms (i.e. have a hapticity of 2 or more). For example, it may bepossible for the ligand of Formula (II) to bind to the metal via the 5contiguous atoms of the imidazolate group (i.e. hapticity of 5, η⁵), asdepicted below in Formula (V).

Accordingly, in some embodiments, the metal ion complex comprises a aligand coordinated to the metal ion via a contiguous series of 2 or moreatoms (i.e. having a hapticity of 2 or more), for example, having ahapticity of 2 (η²), 3 (η⁵), 4 (η⁴) or 5 (η⁵).

The metal ion complexes described herein may form salts. Although thesalts are preferably pharmaceutically acceptable, as discussed below, itwill be appreciated that non-pharmaceutically acceptable salts of themetal ion complexes also fall within the scope of the present invention.Non-pharmaceutically acceptable salts may be useful as intermediates inthe preparation of pharmaceutically acceptable salts (for example, byion exchange such as ion exchange chromatography and/or precipitation,flocculation etc.).

For therapeutic applications of the metal ion complexes, the salts arepreferably pharmaceutically acceptable, that is, a salt which is notdeleterious to a subject to whom the salt is administered. Examples ofpharmaceutically acceptable salts include salts of pharmaceuticallyacceptable cations such as sodium, potassium, lithium, calcium,magnesium, ammonium and alkylammonium; acid addition salts ofpharmaceutically acceptable inorganic acids such as hydrochloric,orthophosphoric, sulphuric, phosphoric, nitric, carbonic, boric,sulfamic and hydrobromic acids; or salts of pharmaceutically acceptableorganic acids such as acetic, propionic, butyric, tartaric, maleic,hydroxymaleic, fumaric, citric, lactic, mucic, gluconic, benzoic,succinic, oxalic, phenylacetic, methanesulphonic,trihalomethanesulphonic, toluenesulphonic, benzenesulphonic, salicylic,sulphanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic,lauric, pantothenic, tannic, ascorbic and valeric acids.Pharmaceutically acceptable salts may also be formed with amino acidshaving positively charged side-chains (e.g. arginine, histidine andlysine), negatively charged side-chains (e.g. aspartic acid and glutamicacid), polar uncharged side-chains (e.g. serine, threonine, asparagineand glutamine), hydrophobic side-chains (e.g. alanine, valine,isoleucine, methionine, phenylalanine, tyrosine and tryptophan) or otherside-chains (e.g. cysteine, selenocysteine, glycine and proline).

The metal ion complexes obtained by the process of the first aspect ofpresent invention and the metal ion complexes of the seventh and eighthaspects of the present invention (including salts thereof, tautomersthereof and polymers thereof) may, in certain circumstances orconditions, aggregate to form a supramolecular assembly. Accordingly, insome embodiments, the present invention provides a supramolecularassembly comprising two or more metal ion complexes of the seventh oreighth aspects of the present invention or obtained by the process ofthe first aspect of present invention. Supramolecular assembliescomprising the metal ion complexes of the present invention may beloosely held together (e.g. by Van der Waals, dispersion orelectrostatic forces) and disassemble under certain conditions.

In addition, some of the metal ion complexes of the present inventionmay form solvates with water or common organic solvents. Such solvatesare encompassed within the scope of the invention.

Pharmaceutical Compositions

The inventors have found that metal ion complexes obtained by theprocess of the first aspect of present invention (including saltsthereof, tautomers thereof and polymers thereof) may have therapeuticactivity. For example, the complex obtained by the process of the firstaspect of the present invention using Cu, EDTA and hydrogen peroxide andallowing the reaction to proceed to completion, and the metal ioncomplexes of the seventh and eighth aspects of the present invention,can be used for reducing inflammation, treating or preventinginflammation associated with arthritis (including osteoarthritis andrheumatoid arthritis), promoting hair growth, treating or preventingpsoriasis, increasing blood flow, treating or preventing chilblains,treating or preventing varicose veins, treating a wound, promoting woundhealing, treating or promoting healing of scar tissue (including scartissue resulting from burns), treating burns, alleviating or reducingjoint pain, alleviating or reducing muscular pain, alleviating orreducing neuropathic or nerve pain, treating or preventing sinusinflammation, treating or preventing sinus pain, treating or preventinga bacterial infection, treating or preventing a fungal infection,treating or preventing eczema, treating or preventing wrinkles, treatingor preventing bruises, treating or preventing joint degeneration,treating or preventing cartilage degeneration, treating or preventingacne, treating or preventing muscle damage, treating or preventing motorneuron disease (MND; MND includes, for example, amyotrophic lateralsclerosis (ALS; also known as Lou Gehrig's Disease), primary lateralsclerosis (PLS), progressive muscular atrophy (PMA), progressive bulbarpalsy (PBP) and pseudobulbar palsy), treating or preventing viralconditions, treating or preventing prion diseases (e.g. bovinespongiform encephalopathy (BSE), scrapie, Creutzfeldt-Jakob disease(CJD) or its variant (vCJD), Gerstmann-Straussler-Scheinker syndrome,fatal familial insomnia or kuru), treating or preventing joint damage,treating or preventing tendon damage, treating or preventingonychoschizia (split nails), treating or preventing cancer, treating orpreventing breast cancer, treating or preventing brain cancer, treatingor preventing melanoma, treating or preventing basal cell carcinoma,treating or preventing squamous cell carcinoma, treating or preventingneuroinflammatory or neurodegenerative diseases (e.g. multiplesclerosis, Parkinson's disease, Alzheimer's disease, dementia,transverse myelitis or epilepsy), treating or preventing asthma or otherbronchial and/or respiratory conditions, treating or preventingosteoporosis, bone fractures or other bone conditions, treating orpreventing eye cataracts or other eye conditions, or combinationsthereof.

The metal ion complex (or mixture of metal ion complexes), or saltthereof, tautomer thereof or polymer thereof, is typically administeredto a patient in the form of a composition comprising the metal ioncomplex (or mixture of metal ion complexes), or salt thereof, tautomerthereof or polymer thereof, and a pharmaceutically acceptable carrier.

Accordingly, the invention also provides a pharmaceutical compositioncomprising a metal ion complex (or mixture of metal ion complexes)obtained by the process of the first aspect of the present invention (ora pharmaceutically acceptable salt thereof, tautomer thereof or polymerthereof) and a pharmaceutically acceptable carrier. Similarly, theinvention also provides a pharmaceutical composition comprising a metalion complex (or mixture of metal ion complexes) of the seventh or eighthaspects of the present invention (or a pharmaceutically acceptable saltthereof, tautomer thereof or polymer thereof) and a pharmaceuticallyacceptable carrier.

In some embodiments, the metal ion complex (or mixture of metal ioncomplexes) of the seventh or eighth aspect of the present invention, orobtained by the process of the first aspect of the present invention (ora pharmaceutically acceptable salt thereof, tautomer thereof or polymerthereof) may be used in combination with one or more othertherapeutically effective agents.

Accordingly, in some embodiments, the pharmaceutical composition mayfurther comprise, or be administered in combination with, one or moreother agents.

It will be understood that the combined administration of a metal ioncomplex (or mixture of metal ion complexes) of the seventh or eighthaspect of the present invention, or obtained by the process of the firstaspect of the present invention, or a pharmaceutically acceptable saltthereof, tautomer thereof or polymer thereof, with the one or more otheragents may be concurrent, sequential or separate administration.

The term “composition” encompasses formulations comprising the activeingredient with conventional carriers and excipients, includingformulations comprising conventional carriers, excipients, creams,lotions etc. used for topical administration. The term “composition”also encompasses formulations with encapsulating materials as a carrierto provide a capsule in which the active ingredient (with or withoutother carriers) is surrounded by the encapsulation carrier. The carrieris “pharmaceutically acceptable” meaning that it is compatible with theother ingredients of the composition and is not deleterious to asubject. The compositions of the present invention may contain otheragents or further active agents as described above, and may beformulated, for example, by employing conventional solid or liquidvehicles or diluents, as well as pharmaceutical additives of a typeappropriate to the mode of desired administration (for example,excipients, binders, preservatives, stabilizers, flavours, etc.)according to techniques such as those well known in the art ofpharmaceutical formulation (See, for example, Remington: The Science andPractice of Pharmacy, 21st Ed., 2005, Lippincott Williams & Wilkins).

The pharmaceutical composition may be suitable for oral, rectal, nasal,topical (including buccal, sub-lingual and slow-release dermal patch),vaginal or parenteral (including intramuscular, subcutaneous andintravenous) administration or in a form suitable for administration byinhalation or insufflation.

The metal ion complex (or a mixture of metal ion complexes) of theseventh and eighth aspects of the present invention (or apharmaceutically acceptable salt thereof, tautomer thereof or polymerthereof), or the metal ion complex (or the mixture of metal ioncomplex(es)) obtained by the process of the first aspect of the presentinvention (or a pharmaceutically acceptable salt thereof, tautomerthereof or polymer thereof), which are sometimes collectively referredto below as the “compounds of the invention”, together with aconventional adjuvant, carrier, or diluent, may thus be placed into theform of pharmaceutical compositions and unit dosages thereof. Thepharmaceutical composition may be a solid, such as a tablet or filledcapsule, or a liquid such as solution, suspension, emulsion, elixir, orcapsule filled with the same, for oral administration. Thepharmaceutical composition may also be in the form of suppositories forrectal administration or in the form of sterile injectable solutions forparenteral (including subcutaneous) use. The pharmaceutical compositionmay also be in a form suitable for metered sterile nasal inhalation,such as a micronized powder diluted in a suitable excipient.

Such pharmaceutical compositions and unit dosage forms thereof maycomprise conventional ingredients in conventional proportions, with orwithout additional active compounds or principles, and such unit dosageforms may contain any suitable effective amount of the active ingredientcommensurate with the intended daily dosage range to be employed.

For preparing pharmaceutical compositions from the compounds of theinvention, pharmaceutically acceptable carriers can be either solid orliquid. Solid form preparations include powders, tablets, pills,capsules, cachets, suppositories, and dispensable granules. A solidcarrier can be one or more substances which may also act as diluents,flavouring agents, solubilisers, lubricants, suspending agents, binders,preservatives, tablet disintegrating agents, or an encapsulatingmaterial.

Suitable carriers include magnesium carbonate, magnesium stearate, talc,sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth,methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoabutter, and the like. Tablets, powders, capsules, pills, cachets, andlozenges can be used as solid forms suitable for oral administration.

Liquid form preparations include solutions, suspensions, and emulsions,for example, water or water-propylene glycol solutions. For example,parenteral injection liquid preparations can be formulated as solutionsin aqueous polyethylene glycol solution.

Sterile liquid form compositions include sterile solutions, suspensions,emulsions, syrups and elixirs. The active ingredient can be dissolved orsuspended in a pharmaceutically acceptable carrier, such as sterilewater, sterile organic solvent or a mixture of both.

The compositions according to the present invention may be formulatedfor parenteral administration (e.g. by injection, for example bolusinjection or continuous infusion) and may be presented in unit dose formin ampoules, pre-filled syringes, small volume infusion or in multi-dosecontainers with an added preservative. The compositions may take suchforms as suspensions, solutions, or emulsions in oily or aqueousvehicles, and may contain formulation agents such as suspending,stabilising and/or dispersing agents. Alternatively, the activeingredient may be in powder form, obtained by aseptic isolation ofsterile solid or by lyophilisation from solution, for constitution witha suitable vehicle, e.g. sterile, pyrogen-free water, before use.

Pharmaceutical forms suitable for injectable use include sterileinjectable solutions or dispersions, and sterile powders for theextemporaneous preparation of sterile injectable solutions. They shouldbe stable under the conditions of manufacture and storage and may bepreserved against oxidation and the contaminating action ofmicroorganisms such as bacteria or fungi.

The solvent or dispersion medium for the injectable solution ordispersion may contain any of the conventional solvent or carriersystems for injectable solutions or dispersions, and may contain, forexample, water, ethanol, polyol (for example, glycerol, propylene glycoland liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils.

Pharmaceutical forms suitable for injectable use may be delivered by anyappropriate route including intravenous, intramuscular, intracerebral,intrathecal, epidural injection or infusion.

Sterile injectable solutions may be prepared by incorporating the activeingredient in the required amount in the appropriate solvent withvarious other ingredients such as those enumerated above, as required,followed by filtered sterilization. Generally, dispersions are preparedby incorporating the various sterilised active ingredient into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, preferredmethods of preparation are vacuum drying or freeze-drying of apreviously sterile-filtered solution of the active ingredient plus anyadditional desired ingredients.

The compounds of the invention may be formulated into compositionssuitable for oral administration, for example, with an inert diluent orwith an assimilable edible carrier, or enclosed in hard or soft shellgelatin capsule, or compressed into tablets, or incorporated directlywith the food of the diet. For oral therapeutic administration, theactive compound may be incorporated with excipients and used in the formof ingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like.

The amount of active compound in therapeutically useful compositionsshould be sufficient that a suitable dosage will be obtained.

The tablets, troches, pills, capsules and the like may also contain oneor more of the components as listed hereafter: a binder such as gum,acacia, corn starch or gelatin; excipients such as dicalcium phosphate;a disintegrating agent such as corn starch, potato starch, alginic acidand the like; a lubricant such as magnesium stearate; and a sweeteningagent such a sucrose, lactose or saccharin may be added or a flavouringagent such as peppermint, oil of wintergreen, or cherry flavouring. Whenthe dosage unit form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier.

Various other materials may be present as coatings or to otherwisemodify the physical form of the dosage unit. For instance, tablets,pills, or capsules may be coated with shellac (or otherwise entericcoated), sugar or both. A syrup or elixir may contain the activecompound, sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye and flavouring such as cherry or orange flavour. Ofcourse, any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic in the amountsemployed. In addition, the active ingredient(s) may be incorporated intosustained-release preparations and formulations, including those thatallow specific delivery of the active ingredient to specific regions ofthe gut.

Aqueous solutions suitable for oral use may include suitable colorants,flavours, stabilising and thickening agents, as desired.

Also included are solid form preparations that are intended to beconverted, shortly before use, to liquid form preparations for oraladministration. Such liquid forms include solutions, suspensions, andemulsions. These preparations may contain, in addition to the activecomponent, colorants, flavours, stabilisers, buffers, artificial andnatural sweeteners, dispersants, thickeners, solubilising agents, andthe like.

For topical administration to the epidermis the compounds of theinvention may be formulated as ointments, creams or lotions, or as atransdermal patch or gels. Ointments and creams may, for example, beformulated with an aqueous or oily base with the addition of suitablethickening, emulsifying and/or gelling agents (e.g. emulsion of oil inwater or emulsion of water in oil). Lotions may be formulated with anaqueous or oily base and will in general also contain one or moreemulsifying agents, stabilising agents, dispersing agents, suspendingagents, thickening agents, colouring agents or preservatives (e.g.methyl paraben, propyl paraben, phenoxyethanol). Other agents may alsobe added to the topical formulations to provide other desiredproperties. For example, humectants, anti-oxidants, vitamins, colouringagents and fragrances may be added. Such additives may be natural (forexample, plant extracts, animal-derived oils, vitamins), or may besynthetic (for example, surfactants or preservatives such as BHT).

As an example, a formulation for topical administration may comprise anyor all of the following: Simmondsia Chinensis (Jojoba oil), glycerylstearate, cetylstearyl alcohol, sodium stearoyl lactylate, stearic acid,glyceryl monostearate, glycerine, caprylic/capric triglyceride, PEG 20,sorbitan monolaurate, cocoa butter, triethanolamine, emu oil, sheabutter, tocopheryl acetate (vitamin E), phenoxyethanol,ethylhexylglycerin, polyoxyethylene (20) sorbitan monooleate,acrylates/C10-30 alkyl acrylate crosspolymer, essential oil (e.g.lavender oil).

Formulations suitable for topical administration in the mouth includelozenges comprising active agent in a flavoured base, usually sucrose,maltitol, acacia or tragacanth; pastilles comprising the activeingredient in an inert base such as gelatin and glycerin or sucrose andacacia; and mouthwashes comprising the active ingredient in a suitableliquid carrier; buccal cavity patches or gels.

Solutions or suspensions for nasal administration may be applieddirectly to the nasal cavity by conventional means, for example with adropper, pipette or spray. The formulations may be provided in single ormultidose form. In the case of a dropper or pipette, this may beachieved by the patient administering an appropriate, predeterminedvolume of the solution or suspension. In the case of a spray, this maybe achieved for example by means of a metering atomising spray pump. Toimprove nasal delivery and retention the compounds of the invention maybe encapsulated with cyclodextrins, or formulated with other agentsexpected to enhance delivery and retention in the nasal mucosa.

The invention also provides use of a metal ion complex (or mixture ofmetal ion complexes) of the seventh or eighth aspect of the presentinvention or obtained by the process of the first aspect of the presentinvention (or a pharmaceutically acceptable salt thereof, tautomerthereof or polymer thereof) in the manufacture of a medicament forreducing inflammation, treating or preventing inflammation associatedwith arthritis (including osteoarthritis and rheumatoid arthritis),promoting hair growth, treating or preventing psoriasis, increasingblood flow, treating or preventing chilblains, treating or preventingvaricose veins, treating a wound, promoting wound healing, treating orpromoting healing of scar tissue (including scar tissue resulting fromburns), treating burns, alleviating or reducing joint pain, alleviatingor reducing muscular pain, alleviating or reducing neuropathic or nervepain, treating or preventing sinus inflammation, treating or preventingsinus pain, treating or preventing a bacterial infection, treating orpreventing a fungal infection, treating or preventing eczema, treatingor preventing wrinkles, treating or preventing bruises, treating orpreventing joint degeneration, treating or preventing cartilagedegeneration, treating or preventing acne, treating or preventing muscledamage, treating or preventing motor neuron disease, treating orpreventing viral conditions, treating or preventing prion diseases,treating or preventing joint damage, treating or preventing tendondamage, treating or preventing onychoschizia (split nails), treating orpreventing cancer, treating or preventing breast cancer, treating orpreventing brain cancer, treating or preventing melanoma, treating orpreventing basal cell carcinoma, treating or preventing squamous cellcarcinoma, treating or preventing neuroinflammatory or neurodegenerativediseases, treating or preventing asthma or other bronchial and/orrespiratory conditions, treating or preventing osteoporosis, bonefractures or other bone conditions, treating or preventing eye cataractsor other eye conditions, or combinations thereof.

The invention also provides a method for reducing inflammation, treatingor preventing inflammation associated with arthritis (includingosteoarthritis and rheumatoid arthritis), promoting hair growth,treating or preventing psoriasis, increasing blood flow, treating orpreventing chilblains, treating or preventing varicose veins, treating awound, promoting wound healing, treating or promoting healing of scartissue (including scar tissue resulting from burns), treating burns,alleviating or reducing joint pain, alleviating or reducing muscularpain, alleviating or reducing neuropathic or nerve pain, treating orpreventing sinus inflammation, treating or preventing sinus pain,treating or preventing a bacterial infection, treating or preventing afungal infection, treating or preventing eczema, treating or preventingwrinkles, treating or preventing bruises, treating or preventing jointdegeneration, treating or preventing cartilage degeneration, treating orpreventing acne, treating or preventing muscle damage, treating orpreventing motor neuron disease, treating or preventing viralconditions, treating or preventing prion diseases, treating orpreventing joint damage, treating or preventing tendon damage, treatingor preventing onychoschizia (split nails), treating or preventingcancer, treating or preventing breast cancer, treating or preventingbrain cancer, treating or preventing melanoma, treating or preventingbasal cell carcinoma, treating or preventing squamous cell carcinoma,treating or preventing neuroinflammatory or neurodegenerative diseases,treating or preventing asthma or other bronchial and/or respiratoryconditions, treating or preventing osteoporosis, bone fractures or otherbone conditions, treating or preventing eye cataracts or other eyeconditions, or combinations thereof, in a human or animal, comprisingadministering to the human or animal a therapeutically effective amountof a metal ion complex (or mixture of metal ion complexes) of theseventh or eighth aspect of the present invention or obtained by theprocess of the first aspect of the present invention (or apharmaceutically acceptable salt thereof, tautomer thereof and polymerthereof).

For example, for treatment or alleviation of inflammation, a topicalformulation (e.g., a cream, lotion, spray or gel) comprising a metal ioncomplex or mixture of metal ion complexes according to the presentinvention may be applied to an inflamed area (e.g. one to three timesper day) until inflammation is reduced. Treatment may continue for aslong as necessary to reduce or continually alleviate inflammation.

As another example, for treatment or alleviation of pain including nerveor neurological pain, a topical formulation (e.g., a cream, lotion,spray or gel) comprising a metal ion complex or mixture of metal ioncomplexes according to the present invention may be applied to a painfularea (e.g. one to three times per day) until the pain is reduced.Treatment may continue for as long as necessary to reduce or continuallyalleviate the pain.

For promoting hair regrowth, a topical formulation (e.g., a cream,lotion, gel, spray or shampoo) comprising a metal ion complex or mixtureof metal ion complexes according to the present invention may be appliedto the scalp (e.g. once or twice per day) for a period of time which isat least sufficient to stimulate or promote hair growth or regrowth.

As another example, for use as a sunscreen, a topical formulation (e.g.,a cream, lotion, gel or spray) comprising a metal ion complex or mixtureof metal ion complexes according to the present invention may be appliedto an area of skin prior to exposure to the sun or UV radiation.Additional applications may be made as required.

As another example, for relief of sinus pain, a topical formulation(e.g., a cream, lotion, gel or spray) comprising a metal ion complex ormixture of metal ion complexes according to the present invention may beapplied to an area of skin around the nose and/or under the eyes oracross the forehead when pain starts, resulting in pain relief.

As another example, to reduce scarring, a topical formulation (e.g., acream, lotion, gel or spray) comprising a metal ion complex or mixtureof metal ion complexes according to the present invention may beapplied, generally one to three times per day, e.g., twice a day, acrossthe scar tissue with rubbing to work it into the scar tissue andsurrounding skin.

As another example, for healing of bruising a topical formulation (e.g.,a cream, lotion, gel or spray) comprising a metal ion complex or mixtureof metal ion complexes according to the present invention may be appliedto and around the bruised area (e.g. once a day) until bruisingdisappears.

As another example, for treatment of onychoschizia (split nails) asuitable formulation (e.g., a liquid, cream, tincture or lotion)comprising a metal ion complex or mixture of metal ion complexesaccording to the present invention may be applied (e.g. once a day)until the desired nail quality is attained.

As another example, for the treatment of skin conditions, such as acne,psoriasis or eczema, a suitable formulation (e.g., a liquid, cream, gel,spray, tincture or lotion) comprising a metal ion complex or mixture ofmetal ion complexes according to the present invention may be applied tothe appropriate area in a manner similar to conventional therapies (e.g.from one to three times per day, and in some cases, twice per day).

As another example, for the treatment of neuroinflammatory conditions,such as multiple sclerosis, Parkinson's disease, Alzheimer's disease,dementia, transverse myelitis or epilepsy, a suitable formulation (e.g.,a liquid, gel, topical spray, nasal spray, dermal or buccal cavitypatch, or sub-lingual lozenge or tablet) comprising a metal ion complexor mixture of metal ion complexes according to the present invention maybe applied to the appropriate area in a manner similar to conventionaltherapies (e.g. from one to three times per day, and in some cases,twice per day).

Dosages

The term “therapeutically effective amount” refers to the amount of acompound that will elicit the biological or medical response in asubject, tissue or cell that is being sought by the veterinarian,medical doctor or other clinician.

It will be understood that the therapeutically effective amount of ametal ion complex will depend upon a variety of factors including theactivity of the specific complex(es) employed, the metabolic stabilityand length of action of that complex, the age, body weight, generalhealth, sex and diet of the subject, the mode and time ofadministration, rate of excretion, drug combinations, and the severityof the particular condition.

It will further be understood that when the complexes of the inventionare to be administered in combination with one or more other agents, orother active agents, the dosage forms and levels may be formulated foreither concurrent, sequential or separate administration or acombination thereof.

EXAMPLES

The present invention is further described below by reference to thefollowing non-limiting Examples.

Materials

Copper turnings: Sigma-Aldrich Pty Ltd, 12 Anella Avenue, Castle Hill,NSW 2154 Australia.

H₂O₂: Redox Pty Ltd, 2 Swettenham Road Minto New South Wales 2566Australia.

Disodium EDTA: Redox Pty Ltd, 2 Swettenham Road Minto New South Wales2566 Australia.

Bulk Density

The metal used in the following Examples (copper) is relatively dense(density of about 8.9 g/cm³). Both “freshly settled” and “tapped” bulkdensities may be determined for the copper particles. The “freshlysettled” bulk density and “tapped” bulk density can be determined byadding 100 g of the copper particles to a 200 ml measuring cylinder. Theinitial volume measured in the measuring cylinder is the “freshlysettled” volume. The measuring cylinder is then picked up and dropped 3times from a height of 2 cm onto a solid surface. The volume measured inthe measuring cylinder at this stage is the “tapped” volume. The“tapped” bulk density can then be determined by the formula:

$\begin{matrix}{{``{tapped}"}\mspace{14mu} {bulk}\mspace{14mu} {density}} \\( {{in}\mspace{14mu} g\text{/}{mL}\mspace{14mu} g\text{/}{cm}^{3}} )\end{matrix} = \frac{100}{{``{tapped}"}\mspace{14mu} {volume}\mspace{20mu} ( {{in}\mspace{14mu} {mL}\mspace{14mu} {or}\mspace{14mu} g\text{/}{cm}^{3}} )}$

For the Examples employing copper wire as the metal particles, there wasno significant difference between the “freshly settled” and “tapped”bulk densities. The bulk density of the copper used in each of theExamples is set out in Table 1. The bulk densities specified in Table 1are the “tapped” bulk density determined as described above.

Example 1

Quantities

Cu (fine wire) 10.0 g H₂O₂ (50 wt. %, aq.) 20.0 g EDTA-Na₂ 44.0 g H₂O80.0 g

Method

Place the Cu fine wire in a 500 ml Pyrex beaker.

Add the EDTA powder to the beaker on top of the Cu fine wire.

Add the water to the beaker.

Add the H₂O₂ to the beaker and swirl to mix.

Results

Experiment continued for 10 days without any noticeable reaction exceptfor some gas release from the base where the metal and undissolved EDTAlay. The liquid was bright blue and became increasingly so over the 10days. Ambient temperature was approx. 20° C. This experiment was allowedto continue for 30 days. No noticeable changes or visible signs ofreaction were observed.

Example 2

Quantities

Cu (fine wire)  7.0 g H₂O₂ (50 wt. %, aq.) 20.0 g EDTA-Na₂ 44.0 g H₂O80.0 g

Method

As per Example 1

Results

Experiment continued for 10 days without any noticeable reaction exceptfor some gas release from the base where the metal and undissolved EDTAlay. The liquid was bright blue and became increasingly so over the 10days. Ambient temperature was approx. 20° C. This experiment was allowedto continue for 30 days. No noticeable changes or visible signs ofreaction were observed.

The reactions in Examples 1 and 2 formed CuEDTA or some other“non-active” metal chelate.

It is believed that this is due to the ambient temperature and the ratioof Cu:EDTA:H₂O₂ not being conducive to the reaction proceeding tocompletion within the time frame used in the Examples.

Example 3

Quantities

Cu (fine wire)  96.0 g H₂O₂ (50 wt. %, aq.) 200.0 g EDTA-Na₂ 438.0 g H₂O800.0 g

Method

Place the Cu fine wire in a 500 ml Pyrex beaker.

Add the EDTA powder to the beaker on top of the Cu fine wire.

Add the water to the beaker.

Add the H₂O₂ to the beaker and swirl to mix.

Results

Ambient temperature approximately 20° C. After 10 days a layer ofapproximately 1 cm of EDTA remains on the bottom of the reaction vessel.Small gas bubbles continue to emanate from the bottom of the reactionvessel seemingly at the metal/liquid interface. The liquid is brightblue. A further 30.0 g 50 wt. aq. H₂O (15 g H₂O₂) was added after 11days.

Still no noticeable change in rate of reaction over the next 48 hours soadded an additional 50.0 g 50 wt. aq. H₂O (25 g H₂O₂). This experimentwas allowed to continue for a further 24 hours.

This last addition of H₂O₂ promoted a significantly more vigorousreaction, but did not result in the “exothermic final reaction”. Theresulting solution was bright blue which we believe is due to thepresence of CuEDTA.

Example 4

Quantities

Cu (fine wire) 100.0 g H₂O₂ (50 wt. %, aq.) 200.0 g EDTA-Na₂ 200.0 g H₂O800.0 g

Method

As per Example 1.

Results

After 12 hours the reaction mixture reached a critical point and anexothermic reaction rapidly ensued, causing the majority of the reactionmixture to “explode” out of the reaction vessel and into the safetycontainer. During this final exothermic reaction, the mixture changed incolour from bright blue to a very dark, not transparent blue/green,“oily” looking liquid.

Of the 100.0 g of Cu starting material, 74.8 g remained unreactedleading to the conclusion that 25.2 g was complexed. Equivalent of 25.2g/L Cu.

Example 5

Quantities

Cu (fine wire) 100.0 g H₂O₂ (50 wt. %, aq.) 200.0 g EDTA-Na₂ 200.0 g H₂O800.0 g

Method

As per Example 1.

Results

Ambient temperatures were in the vicinity of 25° C.-30° C. During thereaction the reaction mixture got very hot. The reaction vessel wasplaced into a controlled temperature water bath at 35° C.

After 11 hours the reaction mixture reached a critical point and anexothermic reaction rapidly ensued and “boiled” over, equally asviolently as Example 4.

Of the 100.0 g of Cu starting material, 69.0 g remained unreactedleading to the conclusion that 31.0 g was complexed. Equivalent of 31.0g/L Cu.

Examples 6 to 15

Method

Used a similar method to that described above in Example 1, but with thereaction mixture heated in a heating mantle to 80° C. The reagents usedare as specified in Table 1.

Results

In each of these Examples, the reaction went rapidly to completion andoverflowed into the capture vessel. For example, in Example 13, thereaction took 13 minutes and considerable steam was released.

Example 16

Quantities

Cu (turnings) 200.0 g (Hot approx. 80° C.) H₂O₂ (50 wt. %, aq.) 132.0 g(Cold) EDTA-Na₂ 220.0 g (Cold) H₂O 390.0 g (90° C.)

Method

Place the Cu fine wire in a 5,000 ml Pyrex beaker and heat to approx.80° C.

Add the EDTA powder to the beaker on top of the Cu turnings.

Add the hot (approx. 90° C.) water to the beaker.

Add the H₂O₂ to the beaker and swirl to mix.

Keep reaction mixture at approx. 80° C. without further agitation.

Results

The reaction went to completion, ending with a strongly exothermicreaction.

Of the 200.0 g of Cu starting material, 169.2 g remained unreactedleading to the conclusion that 30.8 g was complexed. Equivalent of 59.0g/L Cu.

This experiment was repeated 4 further times with equivalent Cucomplexed of 60.1 g/L, 58.4 g/L, 57.2 g/L and 58.5 g/L. Average 58.6g/L.

Example 17 Quantities

Cu (turnings) 200 g H₂O₂ (50 wt. %, aq.) 132 g EDTA-Na₂ 220 g H₂O 390 g

Method

Place the Cu turnings in a Pyrex reaction vessel over a heating mantle.

Dissolve EDTA-Na₂ in water at 80° C. and decant the supersaturatedsolution of EDTA-Na₂ and add that solution to the Cu turnings.

Add the H₂O₂ to the reaction vessel and swirl to mix.

Keep reaction mixture at approx. 80° C. without further agitation.

Results

The reaction did not proceed to a strong exothermic stage, insteadreaching only a weakly exothermic stage. The resulting mixture was clearblue, indicating the likely presence of CuEDTA.

Example 18

Quantities

Cu (fine wire) 1061.6 g H₂O₂ (50 wt. %, aq.)  700.6 g EDTA-Na₂ 1167.7 gH₂O 2070.1 g

Method

Place the Cu fine wire in an oven and heat to approx. 80° C.

Add the EDTA powder to the beaker on top of the Cu fine wire.

Add the hot (approx. 85° C.) water to the beaker.

Add the H₂O₂ to the beaker and swirl to mix.

Keep reaction mixture at approx. 85° C. without further agitation.

Results

The reaction went to completion, ending with a strongly exothermicreaction.

Of the 1061.6 g of Cu starting material, 720.0 g remained unreactedleading to the conclusion that 341.6 g was complexed. Equivalent of47.98 g/L Cu.

In some of the Examples described above a red precipitate (presumably anoxide of copper) was observed to settle to the bottom of the reactionvessel and/or adhere to the walls of the vessel (and occurred bothimmediately and over time). This is easily separated and it is notapparent that this changes the activity of the desired copper ioncomplex.

TABLE 1 Summary of Results Solid Bulk 50 wt. % Cu:EDTA:H₂O₂ EDTA inExample Cu density EDTA aq. H₂O₂ (in mol contact with No. [mol] Cu Type(g/cm³) [mol] [mol] equivalents) Cu 1  10 g Fine Wire 1  44 g  20 g/10 g1.33:1:2.49 yes  [0.157] [0.118]  [0.294] 2  7 g Fine Wire 1  44 g  20g/10 g 0.93:1:2.49 yes  [0.110] [0.118]  [0.294] 3  96 g Fine Wire 1 438g 280 g/140 g 1.29:1:3.52 yes [1.51] [1.17] [4.12] 4 100 g Fine Wire 1200 g 200 g/100 g 2.94:1:5.51 yes [1.57] [0.534] [2.94] 5 100 g FineWire 1 200 g 200 g/100 g 2.94:1:5.51 yes [1.57] [0.534] [2.94] 6 100 gFine Wire 1 250 g 200 g/100 g 2.35:1:4.40 yes [1.57] [0.668] [2.94] 7100 g Coarse 2 200 g 200g/100g 2.94:1:5.51 yes [1.57] Wire [0.534][2.94] 8  40 g Coarse 2 200 g 200 g/100 g 1.18:1:5.51 yes  [0.630] Wire[0.534] [2.94] 9 200 g Coarse 2 1000 g  1000 g/500 g  1.18:1:5.51 yes[3.15] Wire [2.67]  [14.70]  10  20 g Coarse 2 100 g 100 g/50 g1.18:1:5.51 yes  [0.315] Wire [0.267] [1.47] 11 41.2 g Coarse 2 219 g100 g/50 g 1.11:1:2.51 yes  [0.648] Wire [0.585] [1.47] 12 100 gTurnings 1.5 110 g 50 g/25 g 5.35:1:2.51 yes [1.57] [0.294]  [0.735] 13100 g Turnings 1.5 110 g 50 g^(§)/17.5 g 5.35:1:1.75 yes [1.57] [0.294] [0.514] 14 200 g Fine Wire 1 220 g 110 g/55 g 5.35:1:2.75 yes [3.15][0.588]  [1.617] 15 600 g Turnings 1.5 110 g 50 g/25 g 32.1:1:2.51 yes[9.44] [0.294]  [0.735] 16 200 g Fine 1.25 220 g 132 g/66 g  5.35:1:3.30yes [3.15] wire & [0.588] [1.94] Turnings (hot) 17 200 g Turnings 1.5220 g 132 g/66 g  5.35:1:3.30 no (EDTA [3.15] [0.588] [1.94] solution)18 1061.6 g Pine Wire 1.5 1167.7 g 700.6 g/350.3 g 5.33:1:3.28 yes[16.71]  [3.14]  [10.3]  Colour of solution at Example [Cu] ^(Δ) HPLCconclusion of No. Temp (g/L) A:B^(†) Completion experiment 1 ambient — —no blue (~20° C.) 2 ambient — — no blue (~20° C.) 3 ambient 35.40 16:84no blue (~20° C.) 4 ambient 25.20 — yes blue/green (~20° C.) (exothermicreaction observed) 5 ~25-35° C. 31.00 — yes blue/green (exothermicreaction observed) 6 heated 34.33 — yes blue/green (~80° C.) (exothermicreaction observed) 7 heated 27.00 — yes blue/green (~80° C.) (exothermicreaction observed) 8 heated 27.00 30:70 yes blue/green (~80° C.)(exothermic reaction observed) 9 heated — 38:62 yes blue/green (~80° C.)(exothermic reaction observed) 10 heated 11.74 52:48 yes blue/green(~80° C.) (exothermic reaction observed) 11 heated 14.40 16:84 yesblue/green (~80° C.) (exothermic reaction observed) 12 heated 62.4025:75 yes blue/green (~80° C.) (exothermic reaction observed) 13 heated58.80 — yes blue/green (~80° C.) (exothermic reaction observed) 14heated 50.00 35:65 yes blue/green (~80° C.) (exothermic reactionobserved) 15 heated 57.60 — yes blue/green (~80° C.) (exothermicreaction observed) 16 heated 58.64 42:58 yes blue/green (~80° C.)(exothermic reaction observed) 17 heated 23.00 15:85 no blue (~80° C.)18 heated 32:68 yes blue/green (~80° C.) (exothermic reaction observed)^(Δ) Concentration of Cu in solution at end of reaction based onrecovered Cu starting material. ^(†)Ratio of A:B as determined by HPLC,where A is the metal ion complex having a retention time of ~8 min and Bis the metal ion complex having a retention time of ~18 min (PhenomenexLuna HILIC analytical column; 3 μm, 2 × 100 mm; 85:15 CH₃CN/ammoniumformate buffer (0.1M, pH 4.3), 0.3 mL/min). ^(§)35 wt. % H₂O₂ used inExample 13. — Indicates the data was not recorded or not applicable.

Example 19—Characterisation of Metal Ion Complexes

A mixture of copper complexes was prepared using the method describedabove in Example 18.

Initial attempts to separate the mixture of copper complexes by HPLC(Biotage Isolera; DIOL 12 g Reverlis column; UV 300 nm; 85:15CH₃CN/citrate buffer (0.1 M, pH 5.50), 30 ml/min) afforded variousfractions containing copper complexes. For example, “Fraction 1”,“Fraction 2” and “Fraction 6” were obtained, which were analysedindependently (see, for example, ¹H NMR spectra in FIGS. 1, 2 and 3).However, the separation proved difficult in practice andcharacterisation was therefore subsequently performed on the mixture ofcopper complexes obtained using the method of Example 18 (withoutpurification).

Example 18 created a mixture of copper complexes presenting as a darkblue/green liquid having a solids content of 42% and pH 6.5 (referred toherein as “Sample 19.1”).

Sample 19.1 was dried (vacuum, 37.5° C., 24 h), affording a solid thatwas dark green and crystalline in appearance (referred to herein as“Sample 19.2”). Sample 19.2 was found to be highly hygroscopic, highlypolar, highly soluble in water, and had limited (if any) solubility inother polar and non-polar solvents (such as ethanol, methanol, butanol,propanol, chloroform, dimethyl sulphoxide, pentane, propane, butane,hexane etc.).

Sample 19.1 and Sample 19.2 were subjected to various characterisationtechniques. The results of some of these characterisation techniques aresummarised below.

Analytical HPLC

Analytical HPLC was performed on Sample 19.1, indicating the presence oftwo major components. Component A (as referred to in Table 1 above) hada retention time of ˜8 min and Component B (as referred to in Table 1above) had a retention time of ˜18 min (Phenomenex Luna HILIC analyticalcolumn; 3 μm, 2×100 mm; 85:15 CH₃CN/ammonium formate buffer (0.1 M, pH4.3), 0.3 mL/min).

Infrared Spectroscopy

An infrared absorption spectrum of a sample obtained from Example 14 wasrecorded on a fourier transform spectrophotometer (Thermo FisherScientific; thin film), which showed peaks at 3250 and 1600 cm,consistent with the presence of amino group(s) and carboxylic acidgroup(s).

¹H NMR

NMR spectra were recorded on a Bruker AV III 600 MHz spectrometerequipped with a BBFO probe. 1- and 2-dimensional NMR experiments wereperformed, including 1H-13C single and multiple bond correlation (HSQCand HMBC experiments, respectively) on three fractions obtained from theHPLC purification described above. ¹H NMR spectra of Fraction 1,Fraction 2 and Fraction 6 are shown in FIGS. 1, 2 and 3, respectively.

The NMR spectra are consistent with functional groups present in theligands of Formula (I) and Formula (II).

Cyclic Voltammetry Analysis

Sample 19.1 was subjected to cyclic voltammetry and differential pulsevoltammetry. The sample was diluted 50 times in acetate buffer (pH 6.1).The Cyclic Voltammetry analysis indicated the copper to be in the +3oxidation state. The cyclic voltammogram trace is shown in FIG. 3, whichshows two reduction peaks and one oxidation peak, consistent with a Cu³⁺ion.

From the analysis of Samples 19.1 and 19.2, it was determined thatSample 19.1 contained copper complexes of Formula (III) and Formula (IV)(i.e. complexes of Formula (III) and Formula (IV) where M is Cu²⁺ and/orCu³⁺).

Example 20—RT² Profiler PCR Array Gene Expression

In this study, 96 genes were profiled on 3 samples (Sample 19.1 and 2controls) with the PAHS-014Z using the RT² Profiler PCR Array GeneExpression kit.

The RT² Profiler PCR Array is a highly reliable and sensitive geneexpression profiling tool for analysing focused panels of genes insignal transduction, biological processes or disease research pathwaysusing real-time PCR. The catalogued RT² Profiler PCR Array contains alist of the pathway-focused genes as well as five housekeeping(reference) genes on the array. In addition, the array contains a panelof controls to monitor genomic DNA contamination (GDC) as well as thefirst strand synthesis (RTC) and real-time PCR efficiency (PPC). TheqPCR Assays used in PCR Arrays are laboratory-verified and optimized towork under standard conditions enabling a large number of genes to beassayed simultaneously.

Cataloged Arrays

-   -   1. Mature RNA was isolated using an RNA extraction kit according        to the manufacturer's instructions.    -   2. RNA quality was determined using a spectrophotometer and was        reverse transcribed using a cDNA conversion kit.    -   3. The cDNA was used on the real-time RT² Profiler PCR Array        (QIAGEN, Cat. no. PAHS-014Z) in combination with RT² SYBR® Green        qPCR Mastermix (Cat. no. 330529).

C_(T) values were exported to a table that was uploaded to the dataanalysis web portal at http://www.qiagen.com/geneglobe. C_(T) valueswere normalized based on an automatic selection from full panel ofreference genes.

The data analysis web portal calculates fold change/regulation using thedelta delta C_(T) method. In this method, delta C_(T) is calculatedbetween the gene of interest (GOI) and an average of reference genes(HKG), followed by delta-delta C_(T) calculations (delta C_(T) (TestGroup) −delta C_(T) (Control Group)). Fold Change is then calculatedusing the formula 2̂(−delta delta C_(T)).

Fold-Change (i.e. 2̂(−delta delta C_(T))) may also be expressed as thenormalized gene expression (2̂(−Delta C_(T))) in the Test Sample dividedby the normalized gene expression (2̂(−Delta C_(T))) in the ControlSample. Fold-Regulation represents fold-change results in a biologicallymeaningful way. Fold-change values greater than one indicate a positive-or an up-regulation, wherein the fold-regulation is equal to thefold-change. Fold-change values less than one indicate a negative ordown-regulation, wherein the fold-regulation is the negative inverse ofthe fold-change.

Results of this study are summarised in Table 2 below.

TABLE 2 Summary of results from RT² Profiler PCR Array Gene Expressionand Associated Diseases **Fold Up/Down Disease Class Associated Examplesof Diseases/Conditions/Functions Associated Gene Regulation AssociatedPathway with Gene/Pathway with Gene/Pathway and/or Disease Class HES5+19.81 Notch Signaling Tissue Regeneration and Muscle and SkinRegeneration, Brain Repair, Tumour Cell Survival Suppression - includingforebrain tumours, Connective Tissue (including ligament and tendon)Regeneration, Cell Survival (Normal Cells), Impact Injuries HMOX1 +12.68Heme Oxygenase Inflammatory Diseases and Osteoarthritis, Burns(including sunburn), Sprains Signaling Conditions, including and StrainsHypoxia Signalling Vascular, Joint and Brain Neuropathic Pain, Hand andFoot Syndrome, Pain from Analgesia/Pain Relief Impact Injuries SERPINE1+7.56 HIF-1 Signaling Inherited Thrombophilia Thromboses including DeepVein Thrombosis, Pulmonary p53 Signaling and Blood Disorders EmbolismHippo Signaling ACSL5 +4.59 PPAR Signaling Brain Function IncreasesBrain Function and Brain Glucose, Brain Cancer Decreases Inflammation,Decreases Oxidative Stress, Mediates Fatty-Acid-Induced Glioma cellgrowth TNF +4.53 TNF Signaling Neuroinflammatory Diseases Alzheimer'sDisease, Parkinsons Disease, ALS, Cancer Cancer, Major Depression,Psoriasis and Inflammatory Inflammatory Diseases Bowel Disease GADD45B+4.51 p38/JNK Signaling Cancer All Cancers - activates Immune Responseto Cancer Inflammatory Diseases Dementia Senescence Age related diseasesincluding senility SQSTM1 +4.16 NF-κB Signaling Cancer All Cancersthrough activation of Stem Cells and Senescence/Neuronal MND, MS,Parkinsons Inflammatory BMP4 +4.12 Hedgehog Signaling Cancer BreastCancer Inhibition and Inhibition of Inflammatory Diseases MetastasisSenescence Acute Inflammatory Lung Conditions including Asthma, COPDAge-related Macular Degeneration (AMD) SORBS1 +3.07 PPAR SignalingTrauma induced damage Muscle, heart, bone, connective tissues includingInsulin Signaling Diabetes and Obesity tendons and ligament damageInsulin Resistance in Type 2 Diabetes FAS −163.83 p53 Signaling CancerGlioblastoma multiforme, lung (adenocarcinoma), colorectal, advancedgastric, pancreatic, ovarian, breast, and prostate. WNT5A −105.13Hedgehog Signaling Cancer All Cancers - promotes motility andproliferation, including Glioblastoma ID1 −70.04 Notch Signaling CancerAll cancers - inhibits DNA binding, promotes chemoresistance NonSmall-Cell-Lung Cancer, Gastric Cancer, Breast Cancer and is a promoterof Metastasis, Prostate Cancer, Melanoma, Glioblastoma, DIPG CEBPD−51.89 JAK/STAT Signaling Cancer Suppresses tumours OLR1 −39.26 PPARSignaling Cancer Non small cell lung cancer, Gastric Cancer, BreastNF-κB Signaling Cancer and is a promoter of Metastiasis, ProstateCancer, Melanoma, Glioblastoma DAB2 −33.73 Wnt Signaling CancerMelanoma, Colorectal, Cervical, Lung and Stomach NeuroinflammatoryDiseases Cancer Multiple Sclerosis, Parkinsons Disease CCND2 −32.95p53/JAK-STAT Cancer Lung cancers, Thyroid Cancers, Adenocarcinomas ofSignaling Cervix, Squamous Cell Carcinoma ACTB −29.26 Rap1 SignalingCancer Colorectal, Ovarian, Endometrial, Testicular, Hippo SignalingBreast, Urothelial, Pancreatic and Liver and Thyroid Hormone LymphomasSignaling Oxytocin Signaling CDKN1B −27.17 ErbB Signaling Cancer Glioma,Melanoma (p27Kip1) HIF-1 Signaling FoxO Signaling PI3k-Akt SignalingPCNA −25.87 RAD6 Signaling Cancer All Cancers CPT2 −24 PPAR SignalingCancer Hepatocellular Carcinomas, Colorectal Cancers MYC −22.57 MycSignaling Cancer Burkitt Lymphoma and Carcinoma of the cervix, colon,breast, lung and stomach CCND1 −22.53 PI3K/AKT Signaling CancerNon-small cell lung cancers, head and neck squamous cell carcinomas,pancreatic carcinomas, bladder cancer, pituitary adenomas, breastcarcinoma, ER+ breast cancer and hormone therapy resistance in breastcancer, B mantle cell lymphoma NQO1 −22.36 NF-κB Signaling Cancer Colon,breast, pancreas, ovaries and thyroid, and AP-1 Signaling melanoma p53BCL2L1 −22.01 Ras signaling Cancer her2-receptor negative breast cancer,colorectal NF-κB Signaling cancer, non-small-cell lung cancer subtypes,PI3K-Akt Signaling adenocarcinoma, squamous cell carcinoma. Jak-STATSignaling Potent inhibitor of cell death. Inhibits activation ofcaspases. Appears to regulate cell death by blocking thevoltage-dependent anion channel (VDAC) by binding to it and preventingthe release of the caspase activator, CYC1, from the mitochondrialmembrane. Also acts as a regulator of G2 checkpoint and progression tocytokinesis during mitosis. EMP1 −21.39 Src Kinase Signaling CancerEndobronchial lipoma and bronchial neoplasm, asthma, PI3K/AKT SignalingLung Conditions non-small-cell lung cancer SLC27A4 −17.72 PPAR SignalingIntestinal and Obesity Insulin Resistance Syndrome, Obesity RelatedConditions BBC3 −17.2 p53 Signaling Cancer Glioma, colorectal, breast,endometrial, skin, ERK Signaling gastric and liver cancers BCL2 −16.51ErbB Signaling Cancer Melanoma, breast, prostate, chronic lymphocyticRas Signaling Psychological Conditions leukemia, and lung cancer;cGMP-PKG Signaling Autoimmune function Schizophrenia; and cAMP SignalingAutoimmunity PI3K-Akt Signaling VEGF Signaling Apoptosis Signaling STAT1−16.48 IFN Signaling Cancer Renal, gastric, cervical, ovarian and breastcancers EGFR −15.22 EGFR Signaling Cancer Glioblastoma multiforme, lung(adenocarcinoma), Skin Conditions colorectal, advanced gastric,pancreatic, ovarian, breast, and prostate. Psoriasis, eczema andatherosclerosis ACSL4 −13.44 PPAR Signaling Cancer Breast cancer - bothtriple and quadruple negative Adipocytokine Neurological Disorders typessignaling Neuronal Development Mental retardation and otherneurodegenerative diseases PTCH1 −12.56 Hedgehog Signaling CancerMedulloblastoma, nevoid basal cell carcinoma syndrome, esophagealsquamous cell carcinoma, trichoepitheliomas, transitional cellcarcinomas of the bladder HPRT1 −11.96 Purine metabolism MetabolicDiseases Obesity, Hyperthyroidism, Hypothyroidism, Diabetes Drugmetabolism - I & II other enzymes **Fold-Change (2{circumflex over ( )}(- Delta Delta CT)) is the normalized gene expression (2{circumflex over( )} (- Delta CT)) in the Test Sample divided the normalized geneexpression (2{circumflex over ( )} (- Delta CT)) in the Control Sample.Fold-Regulation represents fold-change results in a biologicallymeaningful way. Fold-change values greater than one indicates apositive- or an up-regulation, and the fold-regulation is equal to thefold-change. Fold-change values less than one indicate a negative ordown-regulation, and the fold-regulation is the negative inverse of thefold-change.

The up- or down-regulation of the genes referred to in Table 2 can beused to treat or prevent diseases in humans or animals where the up ordown regulation of the gene would be beneficial. For example, the coppercomplex up-regulates the HES5 gene +19.81 fold. The up-regulation ofthis gene would be expected to promote tissue regeneration, improvinghealing time etc. Similarly, the copper complex significantlydown-regulates the FAS gene (−163.83), a gene implicated in theprogression of cancers.

The copper complexes of Sample 19.1 mimic the activities of the humanGHK-Cu tripeptide, but with advantages over both the native GHKTripeptide and the GHK-Cu Tripeptide. For example, Pickart et al.(Oxidative Medicine and Cellular Longevity; Volume 2012, Article ID324832, doi: 10.115/2012/324832) report that in equivalent studies usingmRNA, GHK showed a 1.9 fold increase in activity over the control,whereas the copper complexes of Sample 19.1 showed a 19.81 fold increasein activity of the HES5 gene. This gene influences tissue regenerationthrough expression of the FGF2 cytokine. This in turn enhances muscleand skin regeneration, brain repair, tumour suppression—includingforebrain tumours, connective tissue (including ligament and tendon)regeneration, and cell survival (normal cells). The implication of thisresult is that this copper complex may provide enhanced fibroblastgrowth over the body's own naturally occurring GHK and GHK-Cu. This isimportant in skin, muscle and other tissue repair. Further, Pickart etal. report that it has not yet been determined whether or not the body'sGHK Tripeptide or GHK-Cu Tripeptide cross the blood brain barrier.However, the inventors have evidence that at least some of the presentcopper complexes may rapidly cross the blood-brain barrier, at a ratethat can be considered as “actively transported” by the body's ownmechanisms. Pickart et al. further report that human GHK Tripeptide doesnot down-regulate any gene pathways, whereas the inventors have foundthat copper complexes of the present invention may significantlydown-regulate, amongst others, the p53 (FAS gene) by ˜160 fold (asmentioned above), the Hedgehog Pathway (WNT5A gene) by ˜105 fold and theNotch (ID-1 gene) by ˜70 fold. The down-regulation of these particularpathway genes has implications for cancer treatment across a range ofcancer types. The inventors have demonstrated anti-cancer activity ofthe copper complexes against glioblastoma, melanoma, breast cancer andbasal cell carcinoma, with treatment showing limited, if any,degradation of normal healthy cells run in parallel. Further, suchcopper complexes up-regulate a number of important regenerative andprotective genes, including the Notch Pathway (HES5 gene; which shows˜20 fold up-regulation), the HO1 Pathway (HMOX1 gene; which shows ˜13fold up-regulation) and the NFkB, PPAR and Hedgehog Pathways (which showan ˜4 fold up-regulation). Together these genes are responsible for(among others): muscle regeneration, brain repair, tumor suppression,tissue repair, cell survival (normal cells), anti-inflammatory activity,anti-oxidant activity, pain relief, TNF activation, brainglucose/function, neuron regeneration, skin regeneration, hair regrowthand bone regeneration.

Example 21—Cell Lines

Sample 19.1 was assessed for activity in various cell lines, as detailedbelow.

Melanoma Cell Line

Sample 19.1 was assayed against the melanoma cell lines MM200 and MelRMuusing a CellTiter 96® AQueous One Solution Cell Proliferation Assay byPromega.

Protocol:

-   -   1. Thaw the CellTiter 96® AQueous One Solution Reagent        (approximately 90 minutes at room temperature, or 10 minutes in        a water bath at 37° C., to completely thaw 20 ml of reagent)    -   2. Pipette 20 μl of CellTiter 96® AQueous One Solution Reagent        into each well of the 96-well assay plate containing the samples        in 100 μl of culture medium.    -   3. Incubate the plate at 37° C. for 1-4 hours in a humidified,        5% CO₂ atmosphere.        -   (Note: To measure the amount of soluble formazan produced by            cellular reduction of MTS, proceed immediately to Step 4.            Alternatively, add 25 μl of 10% SDS to each well to stop the            reaction in order to measure absorbance at a later stage;            SDS-treated plates may be stored protected from light in a            humidified chamber at room temperature for up to 18 hours.)    -   4. Record the absorbance at 490 nm using a 96-well plate reader.

The results of the assay against MM200 cells are summarised in the graphshown in FIG. 5 and the results of the assay against MelRMu cells aresummarised in the graph shown in FIG. 6.

These results demonstrate that at a concentration of 0.1 mg/ml (˜44.3micro molar) 50.8% and 73.3% respectively of the melanoma cells werekilled after 48 hours. These results demonstrate that the copper complexmixture has activity against melanoma cell lines.

Breast Cancer Cell Line

Sample 19.1 was assayed against the breast cancer cell lines MCF7(luminal/estrogen-positive breast cancer cells) and MDA-MB-231(basal/hormone independent breast cancer cells) using a CellTiter 96®AQueous One Solution Cell Proliferation Assay by Promega.

Protocol:

-   -   1. Thaw the CellTiter 96® AQueous One Solution Reagent        (approximately 90 minutes at room temperature, or 10 minutes in        a water bath at 37° C., to completely thaw 20 ml of reagent).    -   2. Pipette 20 μl of CellTiter 96® AQueous One Solution Reagent        into each well of the 96-well assay plate containing the samples        in 100 μl of culture medium.    -   3. Incubate the plate at 37° C. for 1-4 hours in a humidified,        5% CO₂ atmosphere.        -   (Note: To measure the amount of soluble formazan produced by            cellular reduction of MTS, proceed immediately to Step 4.            Alternatively, add 25 μl of 10% SDS to each well to stop the            reaction in order to measure absorbance at a later stage;            SDS-treated plates may be stored protected from light in a            humidified chamber at room temperature for up to 18 hours.)    -   4. Record the absorbance at 490 nm using a 96-well plate reader.

The results of the assay against MCF7 cells are summarised in the graphshown in FIG. 7 a/b and the results of the assay against MDA-MB-231cells are summarised in the graph shown in FIG. 8 a/b.

These results demonstrate that the LD50 for the copper complex mixtureagainst MCF7 is ˜250 micromolar and the LD50 against MDA-MB-231 cells is˜2.2 millimolar. These results demonstrate that the copper complexmixture has activity against breast cancer cell lines.

Human Glioblastoma Cell Line

Sample 19.1 was assayed against the human glioblastoma cell lines U87MGand T98G (using Primary Foetal Astrocytes as control) using thefollowing protocol:

-   -   Cells were seeded in 96 well plates the day before treatment at        pre-optimized concentrations (1.5×10̂4 cells per 96 well plate        for T98G, U87MG, and primary astrocytes).    -   Cells were then treated with varying concentrations of Sample        19.1 (0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.5 mg/ml), and a final        concentration of 1× Real Time Glo reagents (Promega).    -   Cells were incubated and readings of cell metabolism taken at 24        and 48 h.    -   Cell metabolism (death) was measured as a % of untreated cells        and blanked against a no cell background control for each        concentration of Sample 19.1 to remove any background        luminescence from the compound.

The results of these assays are summarised in the graph shown in FIG. 9.

The results of these assays demonstrate that after 48 hours at aconcentration of 0.1 mg/ml (˜44.3 micro molar) only 9.8% of theglioblastoma cells survived whilst 68.0% of the Primary FoetalAstrocytes survived. These results demonstrate that the copper complexmixture has activity against human glioblastoma cell lines.

Example 22

Copper ion complexes prepared by the method described above in Example18 (using copper, EDTA and hydrogen peroxide) were formulated into twotopical formulations. The first formulation was prepared as a cream.This formulation was an oil-in-water emulsion containing 0.95% w/w ofthe copper complex (on a dry weight basis). The second formulation wasprepared as a lotion, which was water based and contained 2.10% w/w ofthe copper complex (on a dry weight basis)

Treatment of Neuropathic Pain

62 year old female suffering long term (5 years) neuropathic pain postchemotherapy and radiation therapy for the treatment of breast cancer.After topical application of the lotion formulation, significant reliefwas experienced for approximately 7 hours. Repeated applicationscontinued to provide significant relief of neuropathic pain.

63 year old male suffering long term (5 years) neuropathic pain postresection of bowel, chemotherapy and radiation therapy for the treatmentof metastatic lung cancer. After topical application of the lotionformulation, significant relief was experienced for approximately 7hours. Repeated applications continued to provide significant relief ofneuropathic pain.

Treatment of Joint Injury

60 year old male suffering damaged shoulder from bicycle accident.

Very limited movement and significant pain. After topical application ofthe cream formulation to the shoulder, immediate pain relief wasexperienced and full range of motion was returned within 7 days(compared to the year predicted by the treating physician).

Treatment of Hand and Foot Syndrome

62 year old male suffering from severe Hand and Foot Syndrome (HFS) as aresult of long term (5 years) chemotherapy and radiation therapy. Aftertopical application of the cream formulation to the affected areas(hands and feet), immediate relief of previously untreatable HFS wasexperienced. Pain was reduced in both hands and feet almost immediately.With longer term use (following treatment 4 times per day for 4 weeks)feeling returned to fingers and hands.

Treatment of Impact Injury

68 year old female had left hand injured by jamming in car door.Immediate high level acute pain, swelling and significant bruisingensued. The lotion formulation was topically applied to the affectedarea immediately after injury, then half hourly for 4 hours then threehourly thereafter. Resulted in immediate pain relief, and overnight, theswelling, pain and bruising completely dissipated.

Treatment of Basal Cell Carcinoma

A 57 year old male with a basal cell carcinoma on his right forearm. Thecream formulation was topically applied to the lesion and an adhesivebandage placed over the carcinoma. Cream was reapplied and the bandagewas changed every 24 hours. The size and appearance of the carcinoma wasvisually examined every 24 hours. Over 3 weeks of treatment, the size ofthe carcinoma reduced and after 3 weeks of treatment, the carcinoma hadbeen completely resolved with no visible scarring.

Treatment of Burns

A 37 year old female with a burn to her left forearm was treated bytopical application of the cream formulation to the burnt areaimmediately after receiving the burn, then as the pain returned over aperiod of 2 weeks. The topical application of the cream formulationresulted in significant pain relief and after 2 weeks of treatment, theburn had healed with no scarring.

It is to be understood that, if any prior art publication is referred toherein, such reference does not constitute an admission that thepublication forms a part of the common general knowledge in the art, inAustralia or any other country.

In the claims which follow and in the preceding description of theinvention, except where the context requires otherwise due to expresslanguage or necessary implication, the word “comprise” or variationssuch as “comprises” or “comprising” is used in an inclusive sense, i.e.to specify the presence of the stated features but not to preclude thepresence or addition of further features in various embodiments of theinvention.

1. A process for the preparation of a metal ion complex, the processcomprising: (a) contacting a metal in the form of particles having abulk density of between about 0.2 and about 8.0 g/cm³ with a chelatingagent in solid form; and (b) while the metal is in contact with thechelating agent, contacting the metal and chelating agent with anoxidising agent, to form at least one metal ion complex.
 2. The processaccording to claim 1, comprising the further step of allowing theoxidising agent, metal and chelating agent to react until completion. 3.The process according to claim 1 or 2, wherein the bulk density of themetal is between about 0.3 and about 4.0 g/cm³.
 4. The process accordingto any one of claims 1 to 3, wherein the bulk density of the metal isbetween about 0.8 and about 2.5 g/cm³.
 5. The process according to anyone of claims 1 to 4, wherein the metal and chelating agent are aboveambient temperature when the metal and chelating agent are contactedwith the oxidising agent.
 6. The process according to any one of claims1 to 5, wherein the oxidising agent is above ambient temperature whenthe metal and chelating agent are contacted with the oxidising agent. 7.The process according to any one of claims 1 to 6, wherein the ratio ofmetal:chelating agent in moles is within the range of about 1:1 to about50:1.
 8. The process according to any one of claims 1 to 7, wherein theratio of chelating agent:oxidising agent in moles is within the range ofabout 1:1 to about 1:20.
 9. The process according to any one of claims 1to 8, wherein the metal is selected from the group consisting of Cu, Zn,Mn, Fe, Co, Ni, Cr, Al, Cd, Ag, Au, Se, and mixtures thereof.
 10. Theprocess according to claim 9, wherein the metal is Cu.
 11. The processaccording to any one of claims 1 to 10, wherein the chelating agentcomprises a nitrogen and/or an oxygen donor.
 12. The process accordingto any one of claims 1 to 11, wherein the chelating agent is selectedfrom the group consisting of ethylenediaminetetraacetic acid (EDTA),diethylenetriaminepentaacetic acid (DTPA),hydroxyethylenediaminetetraacetic acid (HEDTA),ethylenediaminedisuccinic acid (EDDS), salicylic acid, acetyl salicylicacid, amino acids, peptides, and salts thereof.
 13. The processaccording to claim 12, wherein the amino acid is selected from the groupconsisting of glycine, histidine, lysine, arginine, cysteine, methionineand salts thereof, or mixtures thereof.
 14. The process according to anyone of claims 1 to 11, wherein the chelating agent isethylenediaminetetraacetic acid (EDTA) or a salt thereof.
 15. Theprocess according to claim 14, wherein the chelating agent isethylenediaminetetraacetic acid (EDTA) disodium salt.
 16. The processaccording to any one of claims 1 to 15, wherein the oxidising agent isselected from the group consisting of peroxides, peracids, ozone andoxidising salts.
 17. The process according to any one of claims 1 to 16,wherein the oxidising agent is in solution.
 18. The process according toclaim 17, wherein the oxidising agent is in aqueous solution.
 19. Theprocess according to claim 16, wherein the peroxide is hydrogenperoxide.
 20. The process according to claim 19, wherein the hydrogenperoxide is in aqueous solution.
 21. The process according to claim 20,wherein the aqueous solution of hydrogen peroxide has a concentration inthe range of about 5 wt. % to about 60 wt. %.
 22. The process accordingto claim 21, wherein the aqueous solution of hydrogen peroxide has aconcentration of about 50 wt. %.
 23. The process according to any one ofclaims 1 to 22, wherein the metal is Cu, the chelating agent isethylenediaminetetraacetic acid (EDTA) disodium salt and the oxidisingagent is hydrogen peroxide.
 24. The process according to claim 23,wherein the hydrogen peroxide is in an aqueous solution, and the ratioof Cu:ethylenediaminetetraacetic acid (EDTA) disodium salt in moles isabout 1:1 to about 50:1 and the ratio of Cu:hydrogen peroxide in molesis about 1:1 to about 1:20.
 25. The process according to claim 23 or 24,wherein the Cu in contact with ethylenediaminetetraacetic acid (EDTA)disodium salt is maintained at above about 75° C. when the Cu and EDTAare contacted with the hydrogen peroxide, the hydrogen peroxide alsobeing at above about 75° C.
 26. The process according to any one ofclaims 1 to 25, further comprising a step of removing unreacted metaland/or unreacted chelating agent and/or unreacted oxidising agent.
 27. Ametal ion complex obtained by the process according to any one of claims1 to 26, or a salt thereof, tautomer thereof or polymer thereof.
 28. Acomposition comprising a metal ion complex or a mixture of metal ioncomplexes obtained by the process according to any one of claims 1 to26, or a salt thereof, tautomer thereof or polymer thereof.
 29. Aformulation for topical administration comprising a metal ion complex ora mixture of metal ion complexes obtained by the process according toany one of claims 1 to 26, or a salt thereof, tautomer thereof orpolymer thereof.
 30. Use of a metal ion complex or a mixture of metalion complexes obtained by the process according to any one of claims 1to 26, or a salt thereof, tautomer thereof or polymer thereof, in themanufacture of a medicament for reducing inflammation, treating orpreventing inflammation associated with arthritis, promoting hairgrowth, treating or preventing psoriasis, increasing blood flow,treating or preventing chilblains, treating or preventing varicoseveins, treating a wound, promoting wound healing, treating or promotinghealing of scar tissue, treating burns, alleviating or reducing jointpain, alleviating or reducing muscular pain, alleviating or reducingneuropathic or nerve pain, treating or preventing sinus inflammation,treating or preventing sinus pain, treating or preventing a bacterialinfection, treating or preventing a fungal infection, treating orpreventing eczema, treating or preventing wrinkles, treating orpreventing bruises, treating or preventing joint degeneration, treatingor preventing cartilage degeneration, treating or preventing acne,treating or preventing muscle damage, treating or preventing motorneuron disease, treating or preventing viral conditions, treating orpreventing prion diseases, treating or preventing joint damage, treatingor preventing tendon damage, treating or preventing onychoschizia (splitnails), treating or preventing cancer, treating or preventing breastcancer, treating or preventing brain cancer, treating or preventingmelanoma, treating or preventing basal cell carcinoma, treating orpreventing squamous cell carcinoma, treating or preventingneuroinflammatory or neurodegenerative diseases, treating or preventingasthma or other bronchial and/or respiratory conditions, treating orpreventing osteoporosis, bone fractures or other bone conditions,treating or preventing eye cataracts or other eye conditions, orcombinations thereof.
 31. A method for reducing inflammation, treatingor preventing inflammation associated with arthritis, promoting hairgrowth, treating or preventing psoriasis, increasing blood flow,treating or preventing chilblains, treating or preventing varicoseveins, treating a wound, promoting wound healing, treating or promotinghealing of scar tissue, treating burns, alleviating or reducing jointpain, alleviating or reducing muscular pain, alleviating or reducingneuropathic or nerve pain, treating or preventing sinus inflammation,treating or preventing sinus pain, treating or preventing a bacterialinfection, treating or preventing a fungal infection, treating orpreventing eczema, treating or preventing wrinkles, treating orpreventing bruises, treating or preventing joint degeneration, treatingor preventing cartilage degeneration, treating or preventing acne,treating or preventing muscle damage, treating or preventing motorneuron disease, treating or preventing viral conditions, treating orpreventing prion diseases, treating or preventing joint damage, treatingor preventing tendon damage, treating or preventing onychoschizia (splitnails), treating or preventing cancer, treating or preventing breastcancer, treating or preventing brain cancer, treating or preventingmelanoma, treating or preventing basal cell carcinoma, treating orpreventing squamous cell carcinoma, treating or preventingneuroinflammatory or neurodegenerative diseases, treating or preventingasthma or other bronchial and/or respiratory conditions, treating orpreventing osteoporosis, bone fractures or other bone conditions,treating or preventing eye cataracts or other eye conditions, orcombinations thereof, in a human or animal, comprising administering tothe human or animal a therapeutically effective amount of a metal ioncomplex or a mixture of metal ion complexes obtained by the processaccording to any one of claims 1 to 26, or a salt thereof, tautomerthereof or polymer thereof.
 32. A metal ion complex comprising a ligandof Formula (I) or Formula (II)

wherein R¹ is H and R² is H or OH, or R¹ and R² together with the carbonatom to which they are attached form a carbonyl (C═O); R³ is H and R⁴ isH or OH, or R³ and R⁴ together with the carbon atom to which they areattached form a carbonyl (C═O); R⁵ is —CH(OR⁷)CH₂OR⁷ or —CH₂CO₂R⁷ and R⁶is absent or H, or R⁵ is H and R⁶ is —CH(OR⁷)CH₂OR⁷ or —CH₂CO₂R⁷; andeach R⁷ is independently absent or H; or a salt thereof, tautomerthereof or polymer thereof.
 33. The metal ion complex according to claim32, wherein the ligand is of Formula (I), or a salt thereof, tautomerthereof or polymer thereof.
 34. The metal ion complex according to claim33, wherein R⁵ is —CH(OR⁷)CH₂OR⁷ or —CH₂CO₂R⁷ and R⁶ is absent or H, ora salt thereof, tautomer thereof or polymer thereof.
 35. The metal ioncomplex according to claim 34, wherein the ligand is of Formula (Ia)

wherein R¹ is H and R² is OH, or R¹ and R² together with the carbon atomto which they are attached form a carbonyl (C═O); R³ is H and R⁴ is OH,or R³ and R⁴ together with the carbon atom to which they are attachedform a carbonyl (C═O); R⁶ is absent or H; and each R⁷ is independentlyabsent or H; or a salt thereof, tautomer thereof or polymer thereof. 36.The metal ion complex according to claim 35, wherein the ligand is ofFormula (Ib)

wherein R⁶ is absent or H; and each R⁷ is independently absent or H; ora salt thereof, tautomer thereof or polymer thereof.
 37. The metal ioncomplex according to claim 32, wherein the ligand is of Formula (II), ora salt thereof, tautomer thereof or polymer thereof.
 38. The metal ioncomplex according to claim 37, wherein the ligand is of Formula (IIa)

wherein each R⁷ is independently absent or H; or a salt thereof,tautomer thereof or polymer thereof.
 39. A metal ion complex comprising:a metal ion; and a ligand; wherein the complex is a complex obtainablefrom the reaction of EDTA and H₂O₂ in the presence of copper and havingan ¹H NMR in D₂O substantially as depicted in FIG. 1, FIG. 2 or FIG. 3.40. The metal ion complex according to any one of claims 32 to 39,comprising Cu²⁺, Cu³⁺ or a mixture of Cu²⁺ and Cu³⁺.
 41. A compositioncomprising a metal ion complex or a mixture of metal ion complexesaccording to any one of claims 32 to 40, or a salt thereof, tautomerthereof or polymer thereof.
 42. A formulation for topical administrationcomprising a metal ion complex or a mixture of metal ion complexesaccording to any one of claims 32 to 40, or a salt thereof, tautomerthereof or polymer thereof.
 43. Use of a metal ion complex or a mixtureof metal ion complexes according to any one of claims 32 to 40, or asalt thereof, tautomer thereof or polymer thereof, in the manufacture ofa medicament for reducing inflammation, treating or preventinginflammation associated with arthritis, promoting hair growth, treatingor preventing psoriasis, increasing blood flow, treating or preventingchilblains, treating or preventing varicose veins, treating a wound,promoting wound healing, treating or promoting healing of scar tissue,treating burns, alleviating or reducing joint pain, alleviating orreducing muscular pain, alleviating or reducing neuropathic or nervepain, treating or preventing sinus inflammation, treating or preventingsinus pain, treating or preventing a bacterial infection, treating orpreventing a fungal infection, treating or preventing eczema, treatingor preventing wrinkles, treating or preventing bruises, treating orpreventing joint degeneration, treating or preventing cartilagedegeneration, treating or preventing acne, treating or preventing muscledamage, treating or preventing motor neuron disease, treating orpreventing viral conditions, treating or preventing prion diseases,treating or preventing joint damage, treating or preventing tendondamage, treating or preventing onychoschizia (split nails), treating orpreventing cancer, treating or preventing breast cancer, treating orpreventing brain cancer, treating or preventing melanoma, treating orpreventing basal cell carcinoma, treating or preventing squamous cellcarcinoma, treating or preventing neuroinflammatory or neurodegenerativediseases, treating or preventing asthma or other bronchial and/orrespiratory conditions, treating or preventing osteoporosis, bonefractures or other bone conditions, treating or preventing eye cataractsor other eye conditions, or combinations thereof.
 44. A method forreducing inflammation, treating or preventing inflammation associatedwith arthritis, promoting hair growth, treating or preventing psoriasis,increasing blood flow, treating or preventing chilblains, treating orpreventing varicose veins, treating a wound, promoting wound healing,treating or promoting healing of scar tissue, treating burns,alleviating or reducing joint pain, alleviating or reducing muscularpain, alleviating or reducing neuropathic or nerve pain, treating orpreventing sinus inflammation, treating or preventing sinus pain,treating or preventing a bacterial infection, treating or preventing afungal infection, treating or preventing eczema, treating or preventingwrinkles, treating or preventing bruises, treating or preventing jointdegeneration, treating or preventing cartilage degeneration, treating orpreventing acne, treating or preventing muscle damage, treating orpreventing motor neuron disease, treating or preventing viralconditions, treating or preventing prion diseases, treating orpreventing joint damage, treating or preventing tendon damage, treatingor preventing onychoschizia (split nails), treating or preventingcancer, treating or preventing breast cancer, treating or preventingbrain cancer, treating or preventing melanoma, treating or preventingbasal cell carcinoma, treating or preventing squamous cell carcinoma,treating or preventing neuroinflammatory or neurodegenerative diseases,treating or preventing asthma or other bronchial and/or respiratoryconditions, treating or preventing osteoporosis, bone fractures or otherbone conditions, treating or preventing eye cataracts or other eyeconditions, or combinations thereof, in a human or animal, comprisingadministering to the human or animal a therapeutically effective amountof a metal ion complex or a mixture of metal ion complexes according toany one of claims 32 to 40 or a salt thereof, tautomer thereof orpolymer thereof.